DK2623604T3 - Adenovirusbaseret biologisk afgivelses- og ekspressionssystem til anvendelse ved behandling af osteoartrose - Google Patents

Adenovirusbaseret biologisk afgivelses- og ekspressionssystem til anvendelse ved behandling af osteoartrose Download PDF

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DK2623604T3
DK2623604T3 DK12000703.4T DK12000703T DK2623604T3 DK 2623604 T3 DK2623604 T3 DK 2623604T3 DK 12000703 T DK12000703 T DK 12000703T DK 2623604 T3 DK2623604 T3 DK 2623604T3
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Brendan Lee
Kilian Guse
Zhechao Ruan
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Description

DESCRIPTION
[0001] The invention relates to the field of genetic engineering and provides an adenoviral-based biological delivery and expression system for use in the treatment of osteoarthritis in human or mammalian joints by long-term gene expression of human or mammalian interleukin-1 receptor antagonist (11-1 Ra) in synovial cells.
[0002] Osteoarthritis (OA) is a degenerative joint disease that occurs in human or mammalian joints and constitutes a severe economical and medical problem (Matthews, G.L., and Hunter, D.J. (2011). Emerging drugs for osteoarthritis. Expert Opin. Emerging Drugs 1-13., Brooks PM. Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implications. Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is the tough connective tissue that covers the ends of bones in joints. It provides for a relatively frictionless, highly lubricated surface between rigid bones and allows for a smooth movement. During OA development, cartilage is partially or completely lost due to abnormal or excessive wearing, which leads to exposed bone ends that rub against each other resulting in inflammation, pain, swelling or loss of mobility. By now, the detailed reasons for the initial cartilage loss that leads to OA are not known, but there is a strong correlation between the incidence and age, obesity and joint overuse such as excessive athletic activity. Accordingly, OA is a major problem not only in humans, but in many mammals, in particular in horses that join in racings and showjumping.
[0003] Especially in horses, OA constitutes a significant problem with a tremendous economic impact. Horses spend almost their entire life on their legs, and those used for athletic purposes additionally undergo excessive training. Consequently, most of the joints in athletic horses are heavily overused which often results in lameness. Lameness accounts for about 70 % of the cases where horses cannot participate in races or show jumping. About 60 % of the cases can be directly linked to OA (Caron JP, Genovese, RL. Principals and practices of joint disease treatment. In: Ross MW And Dyson S, eds. Diagnosis and Management of Lameness in the Horse. 1st ed. Philadelphia: Saunders, 2003:746-764). Therefore, OA is the most common reason for the inability of a horse to participate in racing competitions or shows.
[0004] No curative treatment is currently available for OA - neither for horses, humans nor for any other mammalian species. Medical treatment is mostly aimed at alleviating the symptoms using analgesic drugs rather than establishing worn away cartilage. An analgesic treatment usually involves steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have shown efficacy in the treatment of OA for some decades. However, while these drugs can suppress joint inflammation, many of them are known to have deteriorating effects on the cartilage, which further worsens the underlying process of OA development. Hyaluronic acid, for instance, which restores viscoelasticity and lubrication of the joints, has also been widely used. Furthermore, polysulphated glycosaminoglycans injected into the joint or intramuscularly as well as orally administered glucosamine and chondroitin sulphate have shown some efficacy, however their mechanisms of action are not fully understood. Thus, currently used therapies have only limited efficacy in the treatment of OAand their success often depends on the severity of the case. Moreover, these drugs must be administered frequently, sometimes even in combination with each other. However, frequent drug injections into the joint are laborious, bear the risk for infections, cause stress for the horse and are costly. In addition, surgery has generally shown low efficacy in horses and is typically only performed in severe advanced-stage subjects. It follows that there is a clear and yet unmet medical need for more efficacious and sustained treatments that are at the same time also cost effective in the long run.
[0005] During OA, interleukin-1 (11-1) functions as a central mediator of inflammation (Dinarello CA. Interleukin-1 family. In: Thomson AW, Lotz MT (eds). The Cytokine Handbook. Academic Press; London, 2003, pp 643-668 .; ). Moreover, 11-1 strongly inhibits matrix synthesis by cartilage and at high concentrations, triggers matrix breakdown (Evans, C.H., Gouze, J.N., Gouze, E., Robbins, P.D., and Ghivizzani, S.C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389 ). To neutralize the effect of 11-1 on synovial inflammation, treatment with interleukin-1 receptor antagonist (11-1 Ra) constitutes a promising concept for treatment of affected osteoarthritic joints (Evans, C.H., Gouze, J.N., Gouze, E., Robbins, P.D., and Ghivizzani, S.C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389.; Caron JP et al. Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression. Arthritis Rheum 1996; 39: 1535-1544). On nucleic acid level, 11-1 Ra is considerably conserved among mammalian species. For example, the cDNA sequences of human II-1Ra (Accession no: NM_173842) shares 82% homology with the murine variant (Accession no: NM_031167), 84% with the equine variant (Accession no: NM_001082525). 84% with the canine variant (Accession no: NM_001003096), 84% with the lapine variant (Accession no: NM_001082770) and 82% with the bovine variant (Accession no: NM_174357).
[0006] The basic concept of using gene therapy for the treatment of arthritis is well established (Evans CH, Robbins PD. Gene therapy for arthritis, In: Wolff JA (ed.). Gene Therapeutics: Methods and Applications of Direct Gene Transform. Birkhauser Boston, 1994, pp 320-343). In the closest prior art, the treatment of equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene using an adenoviral-mediated gene transfer has been described (D.D. Frisbie, S.C. Ghivizzani, P.D. Robbins, C.H. Evans, C.W. Mcllwraith, Gene Ther 9, 12-20 (2002 ). The adenoviral vector used for expression of equine 11-1 Ra DNA was a first-generation adenoviral vector, which was shown to produce biologically active equine 11-1 Ra. Although clinical examinations of the horses in this study indicated that the therapeutic expression of 11-1 Ra significantly decreased signs of joint pain as measured by the degree of lameness, the effect of delivery and expression of biologically active equine 11-1 Ra transgene was only short-term. Already 30 days following treatment of horses with equine 11-1 Ra by intra-articular injection of various amounts of the vector bearing 111-Ra, expression of equine 11-1 Ra in joints dropped to normal levels. Similar results were also detected in the US 2003/0091536 A1, which describes adenovirus particles encoding an interleukin-1 receptor antagonist for use in the treatment of joint disease. The adenovirus particles used were first generation adenoviral vectors.
[0007] Helper-dependent adenoviruses (HDAd), also known as gutless or high-capacity adenoviruses, are the latest generation of adenoviral vectors (Mitani, K., Graham, F.L., Caskey, C.T. & Kochanek. S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci USA 92, 3854-3858 (1995): Parks, R.J. et al. A Helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 93, 13565-13570 (1996); Parks, R.J. Improvements in adenoviral vector technology: overcoming barriers for gene therapy. Clin. Genet. 58, 1-11 (2000)). These vectors are devoid of all viral sequences and are able to mediate long-term gene expression in various tissues (e.g. 7 years in the liver) in contrast to the more immunogenic first generation adenoviruses (Brunetti-Pierri, N. et al. Multi-Year Transgene Expression in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors. American Society of Gene & Cell Therapy, Annual Meeting 2011 Molecular Therapy Volume 19. Supplement 1, May 2011). However, longevity of helper-dependent adenoviruses mediated gene expression in joints has not been evaluated to date.
[0008] The patent US 5,747,072 A describes and claims a recombinant adenoviral vector having an expression control sequence operatively linked to a gene that encodes an anti-inflammatory polypeptide, ribozyme or antisense RNA molecule. Administering to the joint a therapeutically effect amount of a recombinant first generation adenoviral vector resulted in a reduced inflammatory response in the joint of the treated subject. Again, as in other studies, the long-term expression of 11-1 Ra was limited.
[0009] It is therefore an object of the present invention to provide an improved delivery and expression system that allows for a long-term expression of biologically active recombinant interleukin-1 receptor antagonist (11-1 Ra) in synovial cells at human or mammalian joints for treatment and prevention of osteoarthritis. The solution for the problem is provided by an adenoviral-based biological delivery and expression system having the features as claimed in claim 1. Preferred embodiments of the invention are subject-matter of the dependent claims.
[0010] The adenoviral-based biological delivery and expression system according to the present invention is based on a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (11-1 Ra), left and right inverted terminal repeats (L ITR and R ITR), adenoviral packaging signal and non-viral, noncoding stuffer nucleic acid sequences. Furthermore, the gene of the human or mammalian interleukin-1 receptor antagonist (II-1Ra) is controlled by an inflammation-inducible promoter. Preferred inflammation-regulated promoters for use in the present invention are NF-κΒ promoter, interleukin 6 (II-6) promoter, interleukin-1 (11-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1a (MIP-1a) promoter, or hybrid constructs of the above. The promoter sequences are upstream of the reading frame of the cloned 11-1 Ra gene. The inflammation-regulated promoter according to the present invention is specifically activated by increased levels of immune stimulatory substances such as lipopolysaccharide (LPS), which is a major component of the outer cell membrane of gram-negative bacteria. During osteoarthritis, a vartiety of immune stimulatory substances and cytokines are released, resulting in high levels of promoter-activating substances. One component, for instance, is NF-κΒ, which regulates the NF-κΒ promoter. Therefore, the release of such osteoarthritic-specific activators allows for the control of gene expression in joints of humans or mammals for treating or preventing an osteoarthritic condition.
[0011] The use of an inflammation-inducible promoter provides for specific control of 11-1 Ra gene expression in osteoarthritic tissue cells. Only cells that are affected by the disease will express and secrete the 11-1 Ra gene product, whereas cells that are not affected remain silent. The use of NF-KB5-ELAM promoter for inflammation-dependent gene expression is most preferred. However, any other inflammation-dependent promoter that result in a specific expression of the 11-1 Ra gene product in osteoarthritic tissue can be used in context of the present invention.
[0012] The helper-dependent adenoviral vector as used in the present invention minimizes immune responses in the host, and confers long-term gene expression of human or mammalian 11-1 Ra in joints that are affected by osteoarthritis.
[0013] To be most effective, the helper-dependent adenoviral vector of the invention is preferably administered in a single injection dose directly into the joints of the osteoarthritic subjects. Following intra-articular injection, the gene of 11-1 Ra is delivered to synovial cells such as synoviocytes. Synovial cells that are affected by inflammation start to produce recombinant II-1Ra protein under the control of the inflammation-inducible promoter such as the NF-κΒ promoter. High amounts of 11-1 Ra are then secreted into the joint space, where 11-1 Ra is able to inhibit inflammation and stop cartilage degradation by blocking the interleukin-1 receptor on the surface of synoviocytes and the cells embedded in the cartilage. Most importantly, high local concentrations of recombinant 11-1 Ra do not show any side effects.
[0014] Therefore, as shown in the examples below, pain, inflammation and cartilage degradation are inhibited effectively using the adenoviral-based biological delivery and expression system according to the present invention. High local and low systemic concentrations of the therapeutic protein 11-1 Ra are achieved, resulting in maximum efficacy in the treatment of OA at no or minimal side effects. It is further exemplified that cells containing the helper-dependent adenoviral vector of the invention are capable to produce recombinant 11-1 Ra for an extended period of at least one year. Consequently, medical and economic burden associated with frequent joint injections that were required in the known short-term treatments will be significantly reduced. In particular, the serious long-term side effects of commonly used steroids can be avoided by using the helper-dependent adenoviral vector of the invention due to its cartilage protective effect. Thus, common complications associated with OA treatment are minimized and joint health will be preserved in the long run resulting in sustained health improvement of the treated animal or human.
[0015] In addition, the inflammation-dependent 11-1 Ra production of the vector of the invention allows for the prevention of the development of an osteoarthritic condition as synovial cells that are infected with the adenoviral vector of the invention remain silent in the absence of immune stimulatory substances that could activate the NF-KB5-ELAM promoter or any other inflammation-dependent promoter. Only if the osteoarthritic condition initiates, the promoter is activated as a result of inflammation and subsequently 11-1 Ra is produced and secreted. Thus, by using the adenoviral delivery and expression system of the invention, this mechanism allows for the prevention of the development of osteoarthritis in an early stage.
[0016] An inflammation-dependent 11-1 Ra production of the vector of the invention can also be viewed as a safety feature to ensure that 11-1 Ra is no longer produced, for example when the osteoarthritic condition is resolved or has disappeared.
[0017] The helper-dependent adenoviral vector as used in the present invention does not carry any viral sequences, except the left and right inverted terminal repeats (ITRs) and the adenoviral packaging signal. Preferred helper-dependent adenoviral vectors to be used in the present invention are those based on the helper virus and helper-dependent backbone system developed by Palmer and Ng (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852.) and Toietta et al (Toietta, G., Pastore, L, Cerullo, V, Finegold, M., Beaudet, A.L., and Lee, B. (2002). Generation of helper-dependent adenoviral vectors by homologous recombination. Mol Ther 5, 204-210.). A preferred adenoviral delivery and expression system according to the present invention comprises a nucleic acid sequence set forth on pages 15-30, or 31-45, of the description, or a biologically effective part thereof. The nucleic acid sequence on pages 15-30 describes a murine helper-dependent adenoviral vector, and the sequence set forth on pages 31-45 describes a equine helper-dependent adenoviral vector, both bearing the murine and equine 11-1 Ra gene, respectively. Preferably, the system of the invention has at least 50%, 60%, 70%, 80%, 90% sequence homology with the vector set forth on pages 15-30, or 31-45, of the description.
[0018] "Biologically effective" in the context of the present invention means that the gene product of the adenoviral delivery and expression system comprises the full or partial polypeptide sequence of 11-1 Ra having the in-joint activity to neutralize the effect of 11-1 on synovial inflammation.
[0019] The helper-dependent adenoviral vector of the invention preferably contains the cDNA sequence of 11-1 Ra that is controlled by the inflammation-inducible promoter. Although 11-1 Ra contains species-specific nucleic acid sequences, the adenoviral vector is able to express interleukin-1 receptor antagonist (11-1 Ra) from any mammalian species or human. Preferably, the cDNA of the mammalian interleukin-1 receptor antagonist (11-1 Ra) used for cloning is a cDNA selected from the group consisting of murine 11-1 Ra, equine 11-1 Ra, canine 11-1 Ra, cat 11-1 Ra, rabbit 11-1 Ra, hamster 11-1 Ra, bovine 11-1 Ra, camel 11-1 Ra or their homologs in other mammalian species.
[0020] In order to monitor the presence of genomic vector sequences in synovial cells, the helper-dependent adenoviral vector according to the invention preferably further comprises a marker gene that is visually or instrumentally detectable. Preferred marker genes are, for instance, green fluorescence protein (GFP) or luciferase enzyme.
[0021] As an example, the nucleic acid sequence of murine 11-1 Ra as used in the present invention is shown in the sequence listing set forth on page 14 of the description. As noted above, any nucleic acid sequence resulting in a biologically active 11-1 Ra protein of any mammalian or human species can be used in the context of the present invention. Furthermore, also conserved nucleic acid sequences encoding for the same amino acids, polypeptide or protein fall under scope of the present invention. As illustrated in the prior art, 11-1 Ra genes of various species share a high homology among each other. Preferably, the helper-dependent adenoviral vector according to the invention contains a nucleic acid sequence (e.g. cDNA) of 11-1 Ra having at least 50 %, 60 %, 80 %, 90 % sequence homology with the nucleic acid sequence shown in SEQ ID NO 1, The invention also comprises biologically active nucleic acid sequences of 111-Ra or fragments thereof beside the full-length nucleic acid sequence of 111 Ra.
[0022] The present invention further comprises a pharmaceutical composition, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (11-1 Ra), left and right inverted terminal repeats (L ITR and R ITR), packaging signal and non-viral, non-coding stuffer nucleic acid sequences, wherein the expression of the human or mammalian interleukin-1 receptor antagonist (11-1 Ra) gene within synovial cells is regulated by an inflammation-inducible promoter, for the treatment or prevention of osteoathritis. Preferred promoters as used in the context of the present invention are NF-κΒ promoter, interleukin 6 (II-6) promoter, interleukin-1 (11-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1a (MIP-1a) promoter, or hybrid constructs of the above.
[0023] It is a great benefit of the present invention that the adenoviral delivery and expression system specifically locates in the joints when administered intra-articularly. Most importantly, no measurable concentration of vector sequences could be deteced in the liver of mice treated with the adenoviral system of the invention. Therefore, 11-1 Ra concentrations are expected to be highest in the joints injected with the vector of the invention while no significant side effects are expected in any other organ.
[0024] The invention will be further illustrated in the examples following below.
Examples: [0025] High levels of 11-1 Ra were measured in supernatants of synovial cells that were infected with a helper-dependent adenoviral vector (HDAd) of the invention. As shown below, the induction of inflammation with lipopolysaccharide (LPS) led to a dramatic increase of 11-1 Ra concentration as compared with uninduced samples. No 11-1 Ra was detected in non-infected samples (mock) or samples infected with a control vector (HDAd-GFP). The experiments demonstrate that cells infected with HDAd-mll-1 Ra can produce high levels of 11-1 -FRA. It further shows that ll-1Ra is efficiently secreted from those cells, and that inflammatory conditions activate the NF-KB5-ELAM promoter leading to increased 11-1 Ra levels.
Production of the helper-dependent adenoviral vector of the invention [0026] Figure 1 shows gene maps of the HDAd vectors of the invention. The full vector sequence is shown in SEQ ID NO 2 OR SEQ ID NO 3. The only difference between the two vectors is that GQ-201 carries the equine variant of 11-1 Ra whereas HDAd-mll-1 Ra has the murine 11-1 Ra variant. Both vectors contain the inflammation inducible NF-KB5-ELAM promoter upstream of the II-1Ra cDNA according to the sequence on page 14 of the description as well as inverted terminal repeats (ITR) and an adenoviral packaging signal. The vectors were cloned by standard digestion/ligation reactions according to the following strategy. The luciferase cDNA in pNifty-luc, a plasmid that contains the luciferase cDNA driven by a NF-KB5-ELAM promoter, was excised with Ncol and Nhel and cDNAs for equine or murine 11-1 Ra were ligated into this position. The NF-KB5-ELAM promoter - murine 11-1 Ra or NF-KB5-ELAM promoter - equine 11-1 Ra cassettes were excised with Notl and Pad or EcoRI and Pad, blunted and inserted into pLPBL shuttle plasmid, which had been linearized with Sail and blunted. The NF-KB5-ELAM promoter - murine 11-1 Ra or NF-KB5-ELAM promoter - equine 11-1 Ra cassettes were then excised with AscI, which flanks both sides of the multiple cloning site, and ligated into AscI linearized ρΔ28 plasmid (Toietta, G., Pastore, L, Cerullo, V, Finegold, M., Beaudet, A.L., and Lee, B. (2002). Generation of helper-dependent adenoviral vectors by homologous recombination. Mol Ther 5, 204-210.), which yielded the genomic plasmids pe28-mll-1Ra and pA28-eqll-1Ra. These plasmids were digested with Pmel in order to linearize the vector, liberate the inverted terminal repeats and excise bacterial resistance genes. Vectors were rescued and amplified as described before using the helper-virus AdNG163R-2 and 116 cell factories (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852 ;Suzuki, M., Cela, R., Clarke, C., Bertin, T.K.. Mourino, S., and Lee, B. (2010). Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories. Hum Gene Ther 21, 120-126.) HDAd mediates long-term marker gene expression in joints [0027] In order to determine long-term gene expression for up to one year in joints, mice were injected intra-articularly with a helper-dependent adenoviral vector of the invention (HDAd) and, for comparison, a first generation adenovirus (Ad) vector expressing firefly luciferase (luc) under the control of a CMV promoter. Luc expression was followed over time using in vivo bioluminescence imaging. Strong initial luc signals were detected three days after injection with both vectors (Figure 2A). Expression decreased with both vectors thereafter and was undetectable after one month with the first generation vector Ad-luc (Figure 2B). However, HDAd-luc luciferase expression stabilized at day 10 and has been at this level for 380 days. HDAd transduces synovial cells following intraarticular injection [0028] To evaluate HDAd transduction in mouse joints in detail, mice were injected intra-articularly with a LacZ expressing HDAd. Strong LacZ expression was seen in the synovium, however, no expression could be observed in chondrocytes (Figure 3). The inventors also analyzed the liver of these animals to assess whether virus escapes from the joints or is spilled during the injection. Most importantly, no detectable vector concentrations over background could be measured by quantitative PCR (data not shown). Therefore, the vector specifically locates in the joints and remains there, which is of great benefit in the treatment or prevention of an osteoarthritic condition since it suggests minimal side effects. HDAd-ll-1Ra infected cells secrete 11-1 Ra [0029] An HDAd expressing 11-1 Ra under the control of the inflammation inducible NF-KB5-ELAM promoter was generated and its functionality was tested in vitro. High levels of 11-1 Ra were measured in the supernatant of HDAd-ll-1 Ra infected cells on day 3 (Figure 4). Induction of inflammation with lipopolysaccharide (LPS) led to a dramatic increase of 11-1 Ra concentration compared with uninduced samples. No 11-1 Ra was detected in non-infected samples (mock) or samples infected with a control vector (HDAd-GFP). HDAd-ll-1 Ra prevents the development of OAin mice [0030] To assess whether an HDAd expressing 11-1 Ra is able to prevent the development of OA, knee joints of mice were injected intra-articularly with HDAd-ll-1 Ra or a GFP expressing control vector (HDAd-GFP). Two days after injection, cruciate ligament transection was performed to induce OA development This osteoarthritis model was developed in Dr. Brendan Lee's research group and validated in several experiments (Ruan, Z., Dawson, B., Jiang M.M., Gannon, F, Heggeness, M., Lee, B. (2012). Quantitative volumetric imaging of murine osteoarthritic cartilage by phase contrast micro-computed tomography, submitted). The model involves transection of anterior and posterior cruciate ligaments of the knee joints, which leads to development of severe OA. Mice were sacrificed one month after OA induction and joints were prepared histologically and stained with Safranin O. The development of OA was scored by a blinded pathologist according to OARSI (Osteoarthritis Research Society International) standard (assignment of scores on a scale of 1-6, 1: no signs of OA at all, 6: maximum OA). HDAd-ll-1Ra treated joints had significantly lower OA scores than HDAd-GFP treated or untreated joints, suggesting that HDAd-111 Ra prevented the development of OA (Figure 5). The control vector HDAd-GFP did not seem to have any effect on the development of OA since the average OA score was comparable to the score of the untreated group. HDAd-mll-Ra treats OAin a murine model of the disease [0031] The efficacy of HDAd-mll-1 Ra in the treatment of OAwas evaluated in the murine disease model described above. The model was used to assess whether HDAd-111-Ra can efficiently treat OA. Therefore, OA was induced by cruciate ligament transection (except in the untransected group) and OAwas allowed to develop for two weeks. HDAd-ll-1 Ra, the control vector (HDAd-GFP) or vehicle was then injected and mice were sacrificed to analyze the joints another six weeks later. HDAd-GFP treated and uninjected mice developed OAto the same extent with an average score of approximately 4.5 (Figure 6A). However, HDAd-ll-1 Ra treated mice had significantly lower OA scores compared with HDAd-GFP and mock treated. No significant difference was found between HDAd-ll-1 Ra and untransected (OA-free) mice suggesting efficient treatment of the disease or its prevention. The inventors further evaluated the joints in this experiment by micro computer tomography (pCT) analysis. This technique combines high resolution (down to 0.5 micron) x-ray CT scanning with phase contrast optics, which enables visualization of cartilage in small animal joints. Three-dimensional reconstruction of joints and computational tissue analysis tools can be used to quantify several cartilage parameters such as volume and surface area. HDAd-ll-1 Ra treated joints demonstrated significantly higher cartilage volume compared with HDAd-GFP and mock treated joints (Figure 6B). No significant difference was seen between the HDAd-ll-1 Ra and untransected (OA-free) groups. Furthermore, cartilage surface area was significantly larger in HDAd-ll-1 Ra treated mice compared with HDAd-GFP and mock groups (Figure 6C), while no significant difference was seen between HDAd-ll-1 Ra and untransected (OA-free) joints.
Figure Legends: [0032]
Figure 1
The Figure shows a basic gene map of the helper-dependent adenoviral vector of the invention. The vector backbone consists of the left and right inverted terminal repeats (ITR), adenoviral packaging signal (ψ) and non-coding, non-viral stuffer sequences (remaining unmarked sequence between ITRs). The cDNAof murine 11-1 Ra is cloned between the viral left and right ITRs of the used adenoviral vector. The gene of 111-Ra is controlled by inflammation-inducible NF-kB5-ELAM promoter.
Figure 2 A. Helper-dependent and first generation adenoviral vectors mediate the same level of marker gene expression. Mice were injected intra-articularly with 108 virus particles (VP) of a luciferase expressing helper-dependent (HDAd-luc) or a respective first generation (Ad-luc) adenoviral vector. Three days later mice were imaged using MS 200 series imaging system (Caliper Life Sciences, Hopkintom MA). Strong bioluminescence signals were detected in the joints injected with both HDAd-luc and Ad-luc adenoviral vector. Both knee joints of four mice per group were injected; representative pictures of two mice of each group are shown. B. Helper-dependent adenoviral vector mediates long-term marker gene expression in joints. Luciferase expression of the mice described in A was followed by repeated bioluminescence imaging and quantified using Living Image 2.5 software (Caliper Life Sciences). Expression decreased and was undetectable by 30 days with the first generation adenoviral vector (Ad-luc). With the helper-dependent adenoviral vector (HDAd-luc) expression also declined but plateaued at 10 days and has been around this level for 380 days.
Figure 3
Helper-dependent adenoviral vector infects synoviocytes efficiently. Mice were injected intra-articularly with 108 VP of a LacZ expressing HDAd. One day later, mice were sacrificed and LacZ staining on sectioned joints was performed. Strong expression (dark blue staining) was seen in the synovium while no staining could be observed in chondrocytes. Right picture is a higher magnification photograph (40x) of the framed area in the left picture (5x).
Figure 4
Cells infected with HDAd-ll-1 Ra produce large amounts of 11-1 Ra. Human embryonic kidney cells (HEK293) were infected with 100 VP/cell of HDAd-ll-1 Ra, HDAd-GFP or mock. Two days later 11-1 Ra ELISA was performed with cell culture supernatant. Concentrations of about 700 pg/ml were measured for HDAd-ll-1 Ra infected cells, whereas no 11-1 Ra was detectable in the supernatant of HDAD-GFP or mock infected cells. To induce an inflammatory reaction, lipopolysaccharides (LPS, 100 ug/ml) were added to half of the samples and 11-1 Ra concentrations were again determined one day later (day 4). Levels in HDAd-ll-1 Ra samples increased to about 1600 pg/ml whereas uninduced cells produced less 11-1 Ra compared to the previous day. No 11-1 Ra expression was detected in any of the control samples (HDAd-GFP and mock).
Figure 5 HDAd-ll-1 Ra prevents the development of OA. Mice were injected intra-articularly into the knee joints with 108 VP of HDAd-ll-1 Ra, HDAd-GFP or mock and OAwas induced by cruciate ligament transduction two days later. Mice were sacrificed after 4 weeks and joints were histologically prepared, sectioned and stained with Safranin O. A blinded pathologist evaluated the level of OA according to OARSI (Osteoarthritis Research Society International) standards (assignment of scores on a scale of 1-6, 1: no signs of OAat all, 6: maximum OA). Mice treated with HDAd-ll-1 Ra had significantly lower OA scores compared with mice treated with HDAd-GFP and mock. (* indicates significant difference: p < 0.05 by one-way ANOVA; n = 10 joints per group).
Figure 6 HDAd-ll-1 Ra efficiently treats OA in mice. A. HDAd-ll-1 Ra treated joints have significantly lower OA scores compared to controls. OAwas induced in mouse knee joints by cruciate ligament transection and the disease was allowed to develop. Two weeks after transection, mice were injected intra-articularly with 108 VP of HDAd-ll-1 Ra, HDAd-GFP or mock. Mice were sacrificed 6 weeks later and joints were histologically prepared, sectioned and stained with Safranin O. A blinded pathologist evaluated the level of OA according to OARSI (Osteoarthritis Research Society International) standard (assignment of scores on a scale of 1-6, 1: no signs of OAat all, 6: maximum OA). Mice treated with HDAd-ll-1Ra had significantly lower OA scores compared with mice treated with HDAd-GFP and mock. No significant difference was found between the HDAd-ll-1Ra group and age matched, untransected (no OA induction) mice. (* indicates significant difference: p < 0.05 by one-way ANOVA; n = 8 joints per group). B. HDAd-ll-1Ra treated joints demonstrate significantly higher cartilage volume compared to controls. Whole knee joints of mice treated the same way as described above were fixed in electron microscopy fixative and embedded in paraffin. Samples were scanned using X-radia microXCT scanner (Xradia, Pleasanton, CA, USA) and was visualized at 4 micron resolution. Computational 3D reconstruction of joints was performed and cartilage volume and surface area were quantified semi-automatically using TRI BON software (RATOC System Engineering Tokyo, Japan). Significantly higher cartilage volume was measured in HDAd-ll-1Ra treated joints in comparison to controls. HDAd-IMRa joints had similar volumes as untransected (healthy) joints. (* indicates significant difference: p < 0.05, one-way ANOVA, n = 6 joints/group). C. HDAd-ll-1Ra treated joints demonstrate significantly larger cartilage surface area compared to controls. Cartilage surface area was measured as described above. HDAd-ll-1Ra treatment resulted in significantly higher cartilage surface area compared to controls. Surface area of HDAd-ll-1Ra treated joints was similar to that of untransected (healthy) controls. (* indicates significant difference: p < 0.05, one-way ANOVA, n = 6 joints/group).
SEQUENCE LISTING
[0033] <110> Baylor College of Medicine <120> Adenoviral-based biological delivery and expression system for use in the treatment of osteoarthritis <130> HDAd <160> 3 <170> Patentln version 3.3 <210> 1 <211> 535
<212> DNA <213> Murine 111 -Ra <400> 1 atggaaatct gctggggacc ctacagtcac ctaatctctc tccttctcat ccttctgttt 60 cattcagagg cagcctgccg cccttctggg aaaagaccct gcaagatgca agccttcaga 120 atctgggata ctaaccagaa gaccttttac ccgagaaaca accagctcat tgctgggtac 180 ttacaaggac caaatatcaa actagaagaa aagatagaca tggtgcctat tgaccttcat 240 agtgtgttct tgggcatcca cgggggcaag ctgtgcctgt cttgtgccaa gtctggagat 300 gatatcaagc tccagctgga ggaagttaac atcactgatc tgagcaagaa caaagaagaa 360 gacaagcgct ttaccttcat ccgctctgag aaaggcccca ccaccagctt tgagtcagct 420 gcctgtccag gatggttcct ctgcacaaca ctagaggctg accgtcctgt gagcctcacc 480 aacacaccgg aagagcccct tatagtcacg aagttctact tccaggaaga ccaat 535
<210> 2 <211> 29612 <212> DNA <213> Murine helper-dependent adenovirus-111-Ra <400> 2 aaacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60 agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120 tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180 ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240 tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300 ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatatttgt ctagggccgc 360 ggggactttg accgtttacg tggagactcg cccaggtgtt tttctcaggt gttttccgcg 420 ttccgggtca aagttggcgt tttgatatca agcttatcga taccgtaaac aagtctttaa 480 ttcaagcaag actttaacaa gttaaaagga gcttatgggt aggaagtagt gttatgatgt S40 atgggcataa agggttttaa tgggatagtg aaaatgtcta taataatact taaatggctg 600 cccaatcacc tacaggattg atgtaaacat ggaaaaggtc aaaaacttgg gtcactaaaa 660 tagatgatta atggagagga tgaggttgat agttaaatgt agataagtgg tcttattctc 720 aataaaaatg tgaacataag gcgagtttct acaaagatgg' acaggactca ttcatgaaac 780 agcaaaaact ggacatttgt tctaatcttt gaagagtatg aaaaattcct attttaaågg 840 taaaacagta actcacagga aataccaacc caacataaaa tcagaaacaa tagtctaaag 900 taataaaaat caaacgtttg cacgatcaaa ttatgaatga aattcactac taaaattcac 960 actgattttg tttcatccac agtgtcaatg ttgtgatgca tttcaattgt gtgacacagg 1020 cagactgtgg atcaaaagtg gtttctggtg cgacttactc tcttgagtat acctgcagtc 1080 ccctttctta agtgtgttaa aaaaaaaggg ggatttcttc aattcgccaa tactctagct 1140 ctccatgtgc tttctaggaa acaagtgtta acccacctta tttgtcaaac ctagctccaa 1200 aggacttttg actccccaca aaccgatgta gctcaagaga gggtatctgt caccagtatg 1260 tatagtgaaa aaagtatccc aagtcccaac agcaattcct aaaaggagtt tatttaaaaa 1320 accacacaca cctgtaaaat aagtatatat cctccaaggt gactagtttt aaaaaaacag 1380 tattggcttt gatgtaaagt actagtgaat atgttagaaa aatctcactg taaccaagtg 1440 aaatgaaagc aagtatggtt tgcagagatt caaagaaaat ataagaaaac ctactgttgc 1500 cactaaaaag aatcatatat taaatatact cacacaatag ctcttcagtc tgataaaatc 1560 tacagtcata ggaatggatc tatcactatt tctattcagt gctttgatgt aatccagcag 1620 gtcagcaaag aatttatagc cccccttgag cacacagagg gctacaatgt gatggcctcc 1680 catctccttc atcacatctc gagcaagacg ttcagtccta cagaaataaa atcaggaatt 1740 taatagaaag tttcatacat taaactttat aacaaacacc tcttagtcat taaacttcca 1800 caccaacctg ggcaatatag tgagacccca tgcctgcaaa aaaaaaaaaa ttagccaggc 1860 atggtagcat gtacctgtag tcccagctac ttgagaggtg aggtgggaaa atcactttag 1920 tgcaggatgt tgaggctgga gtgaactgtg attgtgccac tgcactccag cctggacaat 1980 agagcaagac cttgtctcaa aaaaatgcat taaaaatttt ttttaaatct tccacgtatc 2040 acatcctttg ccctcatgtt tcataaggta aaaaatttga taccttcaaa aaaaccaagc 2100 ataccactat cataattttt tttaaatgca aataaaaaca agataccatt ttcacctatc 2160 agactggcag gttctgatta aatgaaattt tctggataat atacaatatt aagagagact 2220 gtagaaactg ggccagtggc tcatgcctgt aatcccagca ctttgggagg ctgggtaaca 2280 tggcgaaccc tgtttctaca aaataaaaat attagctggg agtggtggcg cacacctata 2340 gtcccagcta ctcaggaggc tgaggtggaa ggatcgcttg aacccaggag gttgagactg 2400 cagtgaactg tgatcattct gctgcactgc accccagcct gggcaacaga gaccttgtct 2460 caaaaaaaaa aaaaaaagag acaaattgtg aagagaaagg tactctcata taacatcagg 2520 agtataaaat gattcaactt cttagaggaa aatttggcaa taccaaaata ttcaataaac 2580 tctttcccct tgacccagaa attccacttg aataaagctg aacaagtacc aaacatgtaa 2640 aagaatgttt cttctagtac agtcggtaag aacaaaatag tgtctatcaa tagtggactg 2700 gttaaatcag ttatggtatc tccataagac agaatgctat gcaaccttta aaatatatta 2760 gatagctcta gacacactaa tattaaaagt gtccaataac" atttaaaact atactcatac 2820 gttaaaatat aaatgtatat atgtactttt gcatatagta tacatgcata ggccagtgct 2880 tgagaagaaa tgtgtacaga aggctgaaag gagagaactt tagtcttctt gtttatggcc 2940 tccatagtta gaatatttta taacacaaat attttgatat tataatttta aaataaaaac 3000 acagaatagc cagacataca atgcaagcat tcaataccag gtaaggtttt tcactgtaat 3060 tgacttaaca gaaaattttc aagctagatg tgcataataa taaaaatctg accttgcctt 3120 catgtgattx agccccagtc cattaccctg tttaggactg agaaatgcaa gactctggct 3180 agagttcctt cttccatctc ccttcaatgt ttactttgtt ctggtcccta cagagtccca 3240 ctataccaca actgatacta agtaattagt aaggccctcc tcttttattt ttaataaaga 3300 agattttaga aagcatcagt tatttaataa gttggcctag tttatgttca aatagcaagt 3360 actcagaaca gctgctgatg tttgaaatta acacaagaaa aagtaaaaaa cctcatttta 3420 agatcttact tacctgtcca taattagtcc atgaggaata aacacccttt ccaaatcctc 3480 agcataatga ttaggtatgc aaaataaatc aaggtcataa cctggttcat catcactaat 3S40 ctgaaaaaga aatatagctg tttcaatgag agcattacag gatacaaaca tttgattgga 3600 ttaagatgtt aaaaaataac cttagtctat cagagaaatt taggtgtaag atgatattag 3660 taactgttaa ctttgtaggt atgataatga attatgtaag aaaacaacag gccgggcggg 3720 ttggttcaca cgtgtaatcc cagcactttg ggaggctgag gcaggcagac tgcctgagct 3780 caggagttcg agaccagcct gggcaacacg gtgaaatccc gtctctacta aaaatacaaa 3840 aaaattagcc gggtgtggtg acacatgcct gtagtcccag ctacttggga ggctgaggca 3900 ggagaatcac ttgaacctgg gaggtgaagg ttgcagtgag ccaagatggc accacttcac 3960 tccagcctgg gaaacagagc aagactctgt ctctgagctg agatggcacc acttcactcc 4020 agcctgggaa acagagcaag actctgtctc aaaaaaaaca aaacacacaa acaaaaaaac 4080 aggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggcgggtgg 4140 atcacctgag gtcaggagtt ccagaccagc cttgtcaaca tggtgaaacc tccccccgcc 4200 gtctctacta aaaatacaaa aattagccag gcgtggtggc aggagcctgt aatcccagct 4260 acttgggagg ctgaggcagg agaatcgctt gtacccagaa ggcagaggtt gcactgagct 4320 gagatggcac cattgcactc cagcctgggg gacaagagcg agatttcgtc tttaaaaaac 4380 aaaaacaaaa caaaaaacca tgtaactata tgtcttagtc atcttagtca agaatgtaga 4440 agtaaagtga taagatatgg aatttccttt aggtcacaaa gagaaaaaga aaaattttaa 4500 agagctaaga caaacgcagc aaaatcttta tatttaataa tattctaaac atgggtgatg 4560 aacatacggg tattcattat actattctct ccacttttga gtatgtttga aaatttagta 4620 aaacaagttt taacacactg tagtctaaca agataaaata tcacactgaa caggaaaaac 4680 tggcatggtg tggtggctca cacttgtaat cccagtgctt tgggaggctg agacaggaga 4740 gttgcttgag gccaggagtt caagaccgac atggggaatg tagcaagacc ccgtccctac 4800 aaaaaacttt gtaaaaattt gccaggtatg gtggtgcata'cctgtagtcc cagctactcg 4860 ggaggcggag gcagaaggaa tcacttgagc ccaggagttt gaggctgcag tgagctacga 4920 tcataccaca gcactccagc gtggacaaca gagtaagacc ctatctcaaa aacaaaacaa 4980 aacaaaacaa acaaaaaaaa ccacaagaaa aactgctggc tgatgcagcg gctcatgcct 5040 gtaatcccag tattttggga ggcccaggtg ggcgtatcac ctgaggtcag gagttagaga 5100 ccagcctggc caacatggtg aaaccccatc tctactaaaa atacaaaatt agccaggcat 5160 gtggcacgcg cctgtagtcc cagttactgg gaggctgaag caggaggatc acctgagccc 5220 gggaggtgga ggttgcagtg agccgagatc acaccactgc actccagcct gggtgacaca 5280 gcaataccct acctcaaaat aaaaaagaaa aagaaaagaa aagttgctgt ccccgctacc 5340 ccaatcccaa atccaaacag cctctctcat ctcacagtaa gggggaaaaa tcacccaaaa 5400 aagctaagtg atcttttgaa aacccaaact cttagaagtc taagattatt atagtcaact 5460 catgaagtgt catcataaaa gatactctaa tattatttaa gtagaaccac atattggttg 5520 tcttggtatg tctagcccct ggcatacaaa atatttaata acactgatat ggtacctgtg 5580 atgtgaaaat gtactatgag tacagcttta taaatactat atatgtacct atatacagaa 5640 aaaaatacaa caaaatcata aaagcactta tctttgaaag aggagttaca gcaattttat 5700 ttagttcttt attgctttgc tatatattct aaattttttt caatgaatat atatcacttt 5760 taaaaaaatt caatggtctt tcttatåaat tatctttggc agcatgcgtt tttatatata 5820 catataaaat gtatgggaaa tttttaaagg atacattaaa ttaaagcaaa atatacaaac 5880 aaaaaatcag aatacaaaaa gataaaaaga ttgggaaggg agggagggag taaggaggaa 5940 gggtgggtgg gtatagagaa atataccaaa taatggtaag aagtggggtc ttgacacttt 6000 ctacactttt tttaaataaa aaaaattttt ttctctctct tttttttttt tagagacgaa 6060 gtctcgctat gttgcccagg ctggtcttga actcctggga tcaagagatc ctcctgcctc 6120 agcctcccaa ggtgcttgga ttacaggtgt gagccaccac gcctggtcac tttctacact 6180 ttaatatata tattttttca ttttcaatgt catttttatt agttaattta taatacccat 6240 tcaccattat attcaaagtc tatttgaaga aataaaccag aaagaatgaa atactctagc 6300 tcacatgcta ttcaatacta aattaccttt caaatcacat tcaagaagct gatgatttaa 6360 gctttggcgg tttccaataa atattggtca aaccataatt aaatctcaat atatcagtta 6420 gtacctattg agcatctcct tttacaacct aagcattgta ttaggtgctt aaatacaagc 6480 agcttgactt ttaatacatt taaaaataca tatttaagac ttaaaatctt atttatggaa 6540 ttcagttata ttttgaggtt tccagtgctg agaaatttga ggtttgtgct gtctttcagt 6600 ccccaaagct cagttctgag ttctcagact ttggtggaac ttcatgtatt gtcaggttgg 6660 cccgtaatac ctgtgggaca acttcagccc ctgtgcacat ggccaggagg ctggttgcaa 6720 acattttcag gtaggtggac caggacatgc ccctggtcat ggccaggtgg aggcatagtg 6780 ctatacagca ggcagaagtc aatattgatt tgtttttaaa gaaacatgta ctactttcat 6840 aagcagaaaa aatttctatt cttgggggåa aagattatgc'cagatcctct aggattaaat 6900 gctgatgcat ctgctaaacc ttcacatatc agaacatatt tactatagaa agaatgaaaa 6960 tgggacattt gtgtgtcacc tatgtgaaca ttccaaaaat attttacaac aactaagtat 7020 tttataaatt ttatgaactg aaatttagtt caagttctag gaaaatacaa accttgctag 7080 atattataaa aatgatacaa tatatattca tttcaggctc atcagaatat atctgttatc 7140 acttgacaag aatgaaaatg caccattttg tagtgcttta aaatcaggaa gatccagagt 7200 actaaaaatg acttcttcct tgaagcttac tcaccaactt cctcccagtt actcactgct 7260 tctgccacaa gcataaacta ggacccagcc agaactccct tgaaatatac acttgcaacg 7320 attactgcat ctatcaaaat ggttcagtgc ctggctacag gttctgcaga tcgactaaga 7380 atttgaaaag tcttgtttat ttcaaaggaa gcccatgtga attctgccca gagttcatcc 7440 cagatatgca gtctaagaat acagacagat cagcagagat gtattctaaa acaggaattc 7500 tggcaatata acaaattgat ttccaatcaa aacagattta cataccatac ttatgtcaag 7560 aagttgtttt gttttattgc atcctagatt ttattttttt gatttatggt ttactttaag 7620 cataaaaaat ttgtcaatac aactcttccc aaaaggcata aacaaaaatt cataaaactt 7680 gcatcacttg agatacttca ggtatgaatt cacaactttg ttacaactta ctatatatat 7740 gcacacatat atatatattt gggtatattg ggggggttct aatttaagaa atgcataatt 7800 ggctatagac agacagttgt cagaacttgg caatgggtac gtgcaggttc attataccaa 7860 gtctacttgt agttgttcaa aatgtatcat aatacaaggc cgggcgaggt cgtcacgcct 7920 gtaatcccag cattttggga ggctaaggca ggaggattgc ttgaggtcag gagtttgtga 7980 ccagcctggg caacagagca agaccctgtc tccaaaaaga aaaaaaataa ttttttacaa 8040 aataaaaaca aaatgtatca tcagacgaaa ttaaataaga ggcaattcat ttaaatgaca 8100 acttttccca gcttgacatt taacaaaaag tctaagtcct cttaattcat atttaatgat 8160 caaatatcaa atactaattt tttttttttt tttttttttg agacggagtc tcgctctgtc 8220 gcccaggctg gagtgcagtg gcgcgatcct ggctcactgc aagctccgcc tcccgggttc 8280 acgccattct cctgcctcag cctcccgagt agctgggatt acagacatgc gccaccacgc 8340 ccggctaatt ttgtattttt agtagagatg gggtttctcc atgttggtca ggctggtctt 8400 gaatttccca cctcaggtga tctgcctgcc tcagcctcac aaagcagtag ctgggactac 8460 aggcacccac caccacactt ggttaattct tttgtatttt ttttgtaaag acgggatttc 8520 accatgttag ccaggatggt ctcgatctcc tgatctcatg atccgcccgc ctcagcctcc 8580 caaagtgctg ggattacagg cgtgagccac cccgcccggc catcaaatac taattcttaa 8640 atggtaagga cccactattc agaacctgta tccttatcac taatatgcaa atatttattg 8700 aatacttact atgtcatgca tactagagag agttagataa atttgataca gctaccctca 8760 cagaacttac agtgtaatag atggcatgac atgtacatga gtaactgtga acagtgttaa 8820 attgctattt aaaaaaaaag acggctgggc gctgtggctc atgcctgtaa tcccagcact 8880 ttgggaggcc aaggcaagtt gatcgctcga ggtcaagagt'tcgagaccag cctggccaac 8940 gtggtaaaac cccgtctcta ctaaaaatac aaaaaaaaaa ttagccaggc atggtggcac 9000 aggcctgtaa tcccagctac tagggaggct gagacatgga gaactgcttg aatccaggag 9060 gcagaggtta cagtgagccg agatcatacc actacactcc agcctgagtg acagagcgag 9120 actcctgtct aaaaaaaaaa aaaaaaaaaa agatacaggt taagtgttat ggtagttgaa 9180 gagagaactc aaactctgtc tcagaagcct cacttgcatg tggaccactg atatgaaata 9240 atataaatag gtataattca ataaatagga acttcagttt taatcatccc aaacaccaaa 9300 acttcctatc aaacaggtcc aataaactca atctctataa gagctagaca gaaatctact 9360 tggtggccta taatcttatt agcccttact tgtcccatct gatattaatt aaccccatct 9420 aatatggatt agttaacaat ccagtggctg ctttgacagg aacagttgga gagagttggg 9480 gattgcaaca tattcaatta tacaaaaatg cattcagcat ctaccttgat taaggcagtg 9S40 tgcaacagaa tttgcaggag agtaaaagaa tgattataaa tttacaaccc ttaaagagct 9600 atagctgggc gtggtggctc atgcctgtaa atcccagcac tttgggaggc tgaggcgggt 9660 ggatcacctg aggccagaag ttcaagacca gcctagccaa catggcgaaa ccctgtctct 9720 acaaaaaata caaaaattag ccgggtgtgg tggcacgtgc ctgtagtccc agttacttgg 9780 gaggccgagg caggagaatc gcttgaacct aggaggtgga ggctgcagtg agccgagatt 9840 gtgccactgc actccacttc agcctgggcg acaagagcaa gactccgtca caaaaaaaaa 9900 aaaaaaaaaa aagcttaaaa tctagtggga aaggcatata tacatacaac taactgtata 9960 gcataataaa gctcataatc tgtaacaaaa tctaattcga caagcccaga aacttgtgat 10020 ttaccaaaaa cagttatata tacacaaaaa gtaaacctag aacccaaagt tacccagcac 10080 caatgattct ctccctaagc agtatcaagt ttaaagcagt gattacattc tactgcctag 10140 attgtaaact gagtaaagga gaccagcacc tttctgctac tgaactagca cagccgtgta 10200 aaccaacaag gcaatggcag tgcccaactt tctgtatgaa tataagttac atctgtttta 10260 ttatttgtga cttggtgttg catgtggtta ttatcaacac cttctgaaag aacaactacc 10320 tgctcaggct gccataacaa aataccacag actgagtgac ttaacagaaa cttatttctc 10380 acagttttgg aggctgggaa gtccaaaatt aaggtacctg caaggtaggt ttcaatctca 10440 ggcctcttct ttggcttgaa ggtcttctaa ctgtgtgctc acatgacctc ttctaacaag 10500 ctctctggtg tctctttttt tttttttttc ttttttgaga cagagtctca ctctgtcacc 10560 caggctggag tacagtggca caatctgggc tcactgcaac ctccaactcc cgggttcaag 10620 tgattctcat gcctcaccct cccgagtagc tcggatgaca ggagcccgct accacaccca 10680 gctaattttt gtatttttag tagagatggt gtttcactac attggccagg ctggtctcaa 10740 actcctgacc tcgtgatcca cccaccttgg cctcccaaag tgctgggatt acaggtgtga 10800 gccactgcgc ccgtcctggt gtcttttcat ataagggcac taatccaatc agacctgggc 10860 ccaaccctcc cgacttcttc taactgtaat taccttccaa aggccctgtc tccaaatacc 10920 atcacactgg gggttaggac ttcaaaaaåg gtatgggggg' ggtgtgggag gacataaatg 10980 ctcagtccat aacaagcacc caacataaaa atggctagaa cagatcacaa aaaaaaggtc 11040 ctgtatggct ttggggaagg gctcaacccc aaaatatctg agagctctgg aggggcctag 11100 aagtggtaaa tgaatgaaaa cgtggttact ctccagatct gcctttccca aatatggcca 11160 ttcttggctg aatcagaaat caaaggacag gttattaatt actagctcta agttacttac 11220 catttgctga gacagttcag aaatctgact gcatctcctc agagatctag aacacagttc 11280 tcaaattcta acttacttgt gatatacttg tgaatgataa aaatcgctac aggtactttt 11340 attaatctga aagagtattg agaaattacc tttcattctg acttttgtct ggaatgaaaa 11400 tcaatacttt tgctataatc gattactgaa ataattttac tttccagtaa aactggcatt 11460 ataatttttt ttaattttta aaacttcata attttttgcc agactgaece atgtaaacat 11520 acaaattact aataattatg cacgtcacat ctgtaataat ggccttcatg taaacatttt 11580 tgtggtttac acataaaatc tctaattaca aagctatatt atctaaaatt acagtaagca 11640 agaaaattaa tccaagctaa gacaatactt gcaacatcaa ttcatcatct gtgacaagga 11700 ctgcttaagt ctctttgtgg ttaaaaagga aaaaaaaaaa aaagacatgt tggccagatg 11760 cggtggctca cacctgtaat cccagcactt tgggaggctg aggtgggcgg atcacccctg 11820 gcctgcccaa catggtgaaa ccccgtctct actaaaaaca caaaaattag ctgggcgtgg 11880 tggcgggcgc ctgtaattcc agctactcgg gaggctgagg caggagaatt gctagaaccc 11940 aggaggcaga gattgcagtg agctgagatt gcaccattgc actacagtct gggcaacaaa 12000 agtgaaactc catcttaaaa aaaaaaagac aatgttcgtg ggtccaaaca agacttaatg 12060 gaagtgagtc taaaaatgag ctatgtgggc caggcgtagt ggctcccacc tgtaatccca 12120 gcactttggg aggccgaagc aggcagatca tgaggtcagg agatggagac catcctggcc 12180 aacacggtga aatcctgtct ctacaaaaat tagctgggcg tggtggtgcc tgcctgtaat 12240 cccagctact cagaaggctc aggcaggaga atcgcttgaa ccagggagtc ggtggctaga 12300 gtgagccgag atttgcatca ctgcactcct gcctggtgac agagcaagac tccatctcaa 12360 aaaaaacaaa caaaaataaa agataaaaat gagctatgtg aattaaaaga ggtataacaa 12420 tagataaacc atattttatt taattcctag taatgagtaa tatttccaaa cttctggaat 12480 gggcagaaat tgctagttgg catattttta ccttttatat rcagatacat taaaattctc 12540 aaaaaaaaac acctcaaagc agatgatccg ccatctcctt ggataatttg tgttaactca 12600 ggataacaga aaaccaaaat tatgagttac tgatgcaata ttcctaaatg taaaaataat 12660 taaagctaat agtagattca tcttccaatt tcatatcagt cttacaaata aactacatat 12720 ataacttgct tgccttccct tctgagggat aaagctgtta gaagaattaa aatcagcatt 12780 cttgactatt caaccaaggg agggataaat tattactcat tctagggaca tgggctcata 12840 actactacat gtgtaaggac atgaatttac ccaatattac aatttttcct tttattagtg 12900 tgtacagtgg aagaatagac atgttcactc tggacaaaaa aaaaattata cttatcagtt 12960 atcagaagca caatgctgaa gacagtagtt ccataacaat'ttgaagtatg tgatcgaact 13020 agtagattat cttagtagta gtgaattatt gtaaatgtta gtaatttggc agccactggg 13080 cagaaaaata agaattgagg ctcaatattg atattaatgg tggtgattga cacataaatt 13140 ttatcaagtc tacacaatat aaaattacag aaaggtagaa gagtatacca gtacaacttc 13200 aacatatctt cactacaagg gagtaaaatg acatggccta gttactatct aatgaactgc 13260 agaaaactaa aagaaaactc caaggcaact cttctctgct gatctggttg gtccttttcc 13320 taccttttgc aatacccaga tacaaacaat ggatagaaaa caaagtagac ttgtagtatg 13380 caggtcacag tgctaaattc acagaaagaa acccctgaac tgaactgctc tatttcctgg 13440 tggtcacaaa gagtaattct ggtttacacc tacagattga tgtcaatcta caccctgttg 13S00 ataacagtgt ggccaaggac aaaaaaaagg tgctccgttt taccaattct gtaaaaaatt 13560 attggcaggg taagctcggc tagggcagga ttacatttct aggactacca tccccgaaat 13620 ttagaagata ttatatccac ataaagcata tctttcacat taatttgcaa aaatctaaaa 13680 gctttttctt agctcaagtg tgtccaagtt taccctggca gtttaaaacg atagttacaa 13740 gcagcatggg ttgtatcaga cacatttgag ggccaatttc atgtaagtga tattgggcaa 13800 gttacttcaa ctatctgtgc ctccaaggtc atactagtgt ttatttacct aaagggtacc 13860 tgttatgtaa ctttagggtg tttacattag ataatgcctg caaaatattt acttcaacgc 13920 ctaaaacata gttaagtatt caataaatac ctactattgt cactactaac ttaaaagttt 13980 agagattaag agcagaatct ggggtgagac aaacttaggt tcaaatccta gtattgttgg 14040 gtaatcttgg gcaagttact taacctctct gatttgtgta atttaaaaaa ttagttaata 14100 tacataacag ggcttagaag agtatctagc acatagcacc atttaagcat ttgttattgc 14160 taacatgcaa acaatttaag ggaaagaaat tttttaaaaa ggaagaggga tttgcaaact 14220 aaaaacaatg agtatcttat gttcaaagaa aactaacaaa cagccagctc tagcaataat 14280 taaattcact atatactggg gcaggcatca caccccaaag ctaaaagcgt ctacctaggc 14340 caggcacggt ggctcatgcc tgtaatccca gcactttggg aagcagaggc gggcagatcg 14400 cttgagctca ggagttcaag accagcctgg acaacatggc aaaacaccat ctctacaaaa 14460 aatacaaata ttaggccggg cgcagtggct cacgcctgta atcccagcac tttgggaggc 14520 caaggcgggt ggatcacctg agatcaggag ttcgagagta gcctggccaa catggtgaaa 14580 cctcgtctct attaaaaata caaaaaatta gccaggcatg gtggcaggcg cctgtaatcc 14640 cagctactca gggggatgag gtaggagaat cgcttgaacc cgggaggcag aggttgcact 14700 gagccgagat catgccactg tactccagcc cgggcaacaa gagcgaaact ccatctcaaa 14760 aaataaataa ataaataaat aaaataaagt acaaatatta gccagggatg gtggtgcgca 14820 cctgtagtcc cagctacttg ggaggctgaa gtgggagaat cccctgagcc tggggagaat 14880 cacccgagcc cgggaagtcg aggctgcagt gagcagtgat tgtgccactg cactccatcc 14940 taggtgacag agtgagaccc tgtctcaaaa aaaagaaatt ggcagaatta agtaagttga 15000 tgtttagaga tgaaaaatca acattttttc ctcagcaactf gaataaaaac aacagccact 15060 accatttttt tgagtaccta tttgtagcct attttttaac tggtattact cgagagagag 15120 agagctaggt tcgagacaga gctccttctc ttaataactg tatgacctag ggtatgtctg 15180 ttagcctctc tgaggcttca aaggttcctc atctgtaaaa tggtaataat cataccattg 15240 ctacagggct gttttgaaga ctaattagga ctatgtaagt aaacatgatg atggctatta 15300 ttactgttcc ccgccagggg ccatgcaagg gttgctgatt cacatagact gtcttataat 15360 cctctcaata actccaagag gtagccagca cctcagatat acataaaatg acttaagccc 15420 agagaggtga agtaagttgc ccacagccac acaactagta aatagcccaa acaagctgga 15480 ttcccagtta gactccgtta atagcactgc tctttacctt aagtcattac aatgcctaat 15540 atgaaataga atcgcttctt tcttagggtt caagtggtta attatttaat gtattcattc 15600 aacaaaccat catcgaggac ctcttacaag ccaagtactg tgctaagtgc tagagttacg 15660 gcggtgattc ctgcccttaa aaagttttag tgggagaaac aacaggtaac caggtcattg 15720 ccaaaacaac aaaaataatc ataataaagc aggctaaagc atatttaact ggccggggtt 15780 ttgactattt tagcaagcat gatcagaacg gttgaggagg gaggccagca gcttggccgg 15840 ttcaacaaac aagaaaaaac cagtgagggt ggagctaaga taccagaggc tgattacggt 15900 taagaatgtt cttgaaggta aggaccagat tctcattttc tatatcctgg ggcatcggtc 15960 agcatggaat ctggattcta gcacatgtga atttcggctt gaaatgacct aatgcctttt 16020 ccctagttcc ttcgtgtgtc aaatacgcat ggttaccgct accagagctg tagtggggct 16080 tcaatgaggc catgagcatc tccataaaga tgaactacag tgtgtgcaaa actaaaggca 16140 aaacctggtc cccacacgcc ctcccaggtg gtcgctttcc gtgccgaggc ccctccagag 16200 gtgccccgag aacctcacca tcgcacccca aacttccagg gaagggcctc tcccgagaaa 16260 gcccccacgc ccccaccccg cgccatcatt cccgaatctg ccctcggccc ctccccgcag 16320 cacgctcgca ggcggcacat gtcaaccaaa acgccatttc caccttctct tcccacacgc 16380 agtcctcttt tcccagggct cccccgagga gggacccacc ccaaaccccg ccattccgtc 16440 ctccctgccg ccctcgcgtg acgtaaagcc gaacccggga aactggccgc ccccgcctgc 16500 ggggttccct gggcccggcc gctctagaac tagtggatcc caattgaagg cctggtctaa 16560 atgactccaa aatcaccact taattcaaga gactgatttc cctgagtcag gccccttaaa 16620 gcagctattt caatgggaca gggaaacaac cctaggatct ggattagaat cacttggggg 16680 ctgccacacc cccagggctc tgatcctgcc cttctcccac acgcacattc acatactgct 16740 gcagtgacct tccatttcta atgggttcct gggccatctg tcaggtatag ggaatggaaa 16800 aggggttggg gaggctctgc ttcagaaagt ttgtgtcagg ggctcccaga gcctccacag 16860 atagatagca ggggtcccca ccctaccatg gcagctataa atgtgatcaa catttattgg 16920 cctaggatac agcagttagc aaaatgcctg atgtagttcc cactccgtgg aggttgcagg 16980 ctagccaaga agtcatgagt tcagcaaccc ttacgcacca gtgggatgag attggaccag 17040 gccgagggta gtcttgggaa cactcagcåt ttgtctgagg'gccagaagag gctgcttgcc 17100 ctcagacagg aggtcagcat ctttattgta gcccatgaca cctctacacc attgctcttc 17160 tggtcttatg gaagacatct ttgggcctga taacagcgga gtctgtgtcc cacttgtcca 17220 ggctggagtg ccacatcagg cacactccag ttgcagggac agcacagaca agtttcagga 17280 aggctggtgg cctccaggag gttaacctta taaggccaga ttgtaaccta gttgaaaaac 17340 atacacatgc catgataata aaagaaccta ggcaccatta caagagaaaa aatcattttt 17400 gtagatacga gcatggattc ttgggtgggt cagacacact gggcttgtgc tctgactgca 17460 ctgtctcccc tacctgacct tgggtaaacc ataagactgc tgcatgactc agtgtccacc 17520 ccaaaaaagt accggtagat attggccaca gtagatatca gctagagtgg actctcatga 17580 caatgagggg agatgtattc cccatcttag gcacctggga ctctaccttc catcttctgc 17640 tccgtgtctc tccatcccca ggctcttcag aactcaggga gtccagaatg tcagctccca 17700 gatttcagcc ttcagaaagg aaacccatta ccgttcagtt gaacaaatgt tgtctgagcc 17760 ccagatctgg gctcagaggc catctaggct atgagacaag aggggaacaa agcaccgtct 17820 gcactcactc accacactca cttgctgtcc caggtcacat ccatcgggta gagaatctaa 17880 gaggctgagc tagctcccgc caccagccca gcccacccca cctggcccct tccttccttc 17940 tacaaaatat gcaccacctg tcaaagggtg ggcagtgcca ggcctgcata cagagcactg 18000 agtgtaaaag cagacatgga ccctgacctc caggagcttc caattttctt gaagagacaa 18060 atcagctggc atttcagtcc agtgtgatct gctcttggtg agcacagacc tagggagttg 18120 gggcagcttc ccagaagaac tgcagtccag gctgagggca gagaaatgag gggaatggcg 18180 aggaattggg gagcaggggg gagctcagta gagagccaag ggcgggaggt gagaagtccg 18240 tgttgggcca ggagctaccc tccggtggcc acagccgaag tcgaggatgc ctttggaact 18300 catccccact tctctctttc tgtatgtagc cgtccaagaa caagtcacct ccaagtgtag 18360 ccggatcaag gcaagccccc catctagcaa gcacttgatg ccacccagaa ctgggcttct 18420 tcagaacaat ctgagtccag gaatgatccc actcaccagg caccagagct gcgagggcat 18480 gggagtgatc tcaccaactc tggggaagcg gcaaggaatt ttcacctcca gcccccagtg 18540 tcccatcctc tcacactcag gccagactcc cctgggcaga cttgactctg tctgccagca 18600 tatgcagagc cccaaggcca ccccaccaga agtgcccctg cctgggttct gtcccagctc 18660 cctgggcacc cagtccttga gtccccacca gctcagacgg cctagtgtgc caagaatgcc 18720 cactgcgttc aacaatgctg catgggtcac agcggcagca gctgtgacca cagcagtttc 18780 ggggaaaaca cccctcagcc aagtggataa tagcgttcag cagcactcac cttctggcca 18840 ggcctgcctt cagaggccat ctgattggga ggcacaagtg cccgctgcga tgggaacaca 18900 agtgcccctg gccaacaacc ccagcttcag cctgctgggc agccagagcc tcaggcagag 18960 cccggtacag ggcccggtgc ctgtagcaaa caccaccaag ttcctccagc agggtatggc 19020 cagctttagt cccctgagcc ccatacaggg catcgagcca ccaagctatg tggctgctgc 19080 tgccaccgct gctgctgctt ctgccgttgc tgccagccag' ttcccaggtc cgttcgacag 19140 aacggatatt ccccctgagc tgccacctgc cgactttttg cgccagcccc aacccccact 19200 aaatgatctg atttcgtcac ctgactgcaa tgaggtagat ttcattgaag ctctcttgaa 19260 aggctcctgt gtgagcccag atgaagactg ggtgtgcaac ttgaggctga tcgacgacat 19320 tttggaacag catgctgctg ctcaaaatgc cacagcccag aattctgggc aagtcaccca 19380 ggatgctggg gcactttaaa tctgagcagg atgcccatag aaacccccat ggtgacatca 19440 ctctaggaag tggtgtcgat ccatacccgc agttgtctcc cgttacaatt tgagtggtgt 19500 tgtcagccca tgcttatccc tctctctacc tgtgacaaaa tggaaagctg gtgatttttc 19560 aagctacgtg tacatatttg aaaattttgt aaatggtttt cctaaacatt aatgacagaa 19620 gtatttatac ttcattttgt gactttgtaa ataaagcgac ggcttttgtt tcagtagagt 19680 tgtgtttact atgcattgtt ttgtgtttat tatacaatgt tacaaatatg cagaccgtgt 19740 tgtttgctcc agtgatacct tgttaagcta ggtggctgag tcgcttatgg ttttaatgca 19800 atgagcaatg tggatatgac caagagttgt tgtgcaagtt gacaaatgcc aaatagaaaa 19860 ccacttggcc atttatttct atgttcacta aaaatcctat tgccttgtgt gattcttaat 19920 ctcttttgcg aacctttcag tctccgctag ctctttccta atgagcttta cagcagaagc 19980 tgttttatcg ttaagtgccc cacagagaca ctttaccagg aggctgggag agttctccag 20040 atttgggaga ggcgcagaga cagtgtgtga gccgagccct gtctcagcaa tccacctgga 20100 ggagctagag tatcctcctc cctttaccat tcagaccgag agaaaaagcc cagcttgtgt 20160 gcaccctcgt ggggttaagg cgagctgttc ctggtttaaa gcctttcagt atttgttttg 20220 atgtaaggct ctgtggtttg ggggggaaca tctgtaaaca ttattagttg atttggggtt 20280 tgtctttgat ggtttctatc tgcaattatc gtcatgtata tttaagtgtc tgttatagaa 20340 aacccacacc cactgtcctg taaacttttc tcagtgtcca gactttctgt aatcacattt 20400 taattgccac ctcgtatttc acctctacat ttgaaatctg gcgtctgttt caagccagtg 20460 tgttttttct tcgttctgta ataaacagcc aggagaaaag tgcctctatg tttttatttt 20520 tcaagggagt attcagtacc tacaaaccca agtcaggaag cctgctagtg gctttggttc 20580 tttcagaggc tgctcgatgc cttgtgtgtc agaaagaaag attcagcagt tttgcatcat 20640 ggcaaagaag cctgttattt tggggctcag ccccrcattt tatagaggat gaaacagagg 20700 gggatgggag gtcacaaaga caactgcccc gggagcaggt gtgggggaga cttgccctga 20760 gggtctagac gctctgcacc accgtcctgt ctcccttgct gaagaccaca catgcccttc 20820 tttgaccaga ccctgccacc tgataggcca ggacctggta ggcgggtacc caggtttcat 20880 ggatggaacc acatctcccc aaaagtgggg aggtagctac tgggatgcac gcctcccgcc 20940 atgtgctata ggagagcagc tgaagcaaca gttgggatca gatgtagtca caattgaatg 21000 catcatcaca tttatccctc taagtggctg ggagagttga tatcctcatc cctaaggtac 21060 aaaatgttcc aatttgatca gtggctttca ggagctgaga aaggcatgtg ctctgaggca 21120 gagctgttat gtcccgcaga gcctaaaaåt gctctaagaa' catgctccct gccaaaattc 21180 tcaatggctg tgacaaggga caacgatcga ccaatggggg tggaagcaga cctccgcagt 21240 ccaggggcca gagctaggac agaggggtcg gagaaagagt cattttccca acactccagc 21300 tcttggccag tcctcacaca gtcccctcct gcttcctgct gagagagata tcctcatagg 21360 tctgggtaaa gtccttcagt cagctttcat tccctgtcac caactttgtc tctgttctcc 21420 ctgcccgtct caggcagcac tcctcaggaa acctctccaa gagccagcct cactgcagcg 21480 cccactattg tccctctgcc tcaagtgtcc catccatgcc aggccccagg caggctgcag 21540 ctttccctca gggccacacc aaagcacttg ggctcagctg tgctgtcccc ctccatcact 21600 gagctcaggg gcagcagggg tggggtgcca ggaggcccat tcacccttct ctggctctgt 21660 gttggaccca cctgcccagc cactgctgct tagaacctac ccgctgggaa aatgaagccc 21720 tcccggaggg gccacctcaa cctgagagcc tcacggatca cagttgtccc cactcagctc 21780 tgccagccct cagagaccca tagataaaag ctgagcttgg ctcgcagagc tggttccatc 21840 ttccattccc agagggttca acttcctacc ccaaccacac agggaacctc aaggctgagc 21900 cagtgtgggc tgcagtgcag accagcttcc tggacacgtc ctgccacctg accccaggct 21960 ggcctcactg cccctggcac tcctgaccct atcctcattc ctcctggcag tgcgtgttct 22020 gccattccgc tttcccttag ctgtcctctc actgtactgt cagcttctcc ttttccaggt 22080 gccccccagg ggctttccac atgaccctgt caccccacag cccatccagc accaattcca 22140 gctctctgcc acccttcaaa ggagtgacag tgccctgctt cacctcccac tcacccctca 22200 acccagagca atctggctcc agtcttgcct ccttccccct aagtactcta gtcacagttc 22260 caaattcctc ctggtcataa agccaaatga agcttcctgg tcctcagcgg acttgccact 22320 tcagcagtac tggactctct cctcccagaa acctgtttcc ccttggctcc tggagcccac 22380 actctgctgg aatccttctg cctctctggc ctgtagcctg gccctctctc ccaacctgag 22440 gtccattctc tcctgctcct ccacaagatg ttgctccttc cattacttcc tccctctcaa 22500 ccaaagctcc ttcattagct ctttatcttc tggtttcttc ccctgggcag acgaatggat 22S60 tcaagagcct gtggcccagc agcccagcac tccaggatct cagcacttca gcatcccagt 22620 accctagcat ctcaataccc cagcacccca gcaccatagt attccagcac cccattgtcc 22680 aagcatctca gcactccagc atcccagcac cccaacactc cagcagccca gaatctcagc 22740 accctagcac tgcagcatct caggacccca gcacttcagc atcccagcac actagtactc 22800 cagcatctcg gcaccccagc acctaggcat cccaacaccc agcaccccag cacttaagca 22860 tcccaccact acagtatctc aacactccag caccccagca ccatagtgtt ccagcacccc 22920 agcatcccaa caccccagca cttaagcatc ccaacacctc ggcatcccaa caccccagca 22980 ctgcagcatc tcagcacctt agcatcccag tgccctagca tctcaatgct ccagcacacc 23040 agtactacag tattccagca ccccagcact ccagcatctc agcactgcag cactgcagca 23100 ctccagcatc ccaaaatccc agcatcccaa caccccagca gaccagcaga ccagcatctc 23160 agcaccgcag catccaagga ctatcccagc atcccagcaa'cccagcacct cagcatccca 23220 acaccccagc atttcagcat ggcaacaccc cagtacccca gcacttcagc accccagtat 23280 cccagcatct cagcgaccca gtatcacaaa acctcagcat cctagcaccc cagcacccca 23340 gcaccttagc accttagcat cccagcatct cagcgcctca gcatcttgat attctggctg 23400 aggtcagcgt ggtgtatcta gtcagggtcc taactttcac ttcgcaggga aatgctgctg 23460 gactgggtct catgttgggc tgaagctctc tagacccctt gaagacagca taaaagagct 23520 tggagacgct gggtgtcccc catggaagag ttcactctca tcctgctttg acaacagcct 23580 tctctggggt ccctcacggg cccctctttc ttactgcaag tttgtctctg agaagactgt 23640 gatgcagaag tcactcagct gcctgtggct cctgaagagc tgaaggtgga ggcctgtagg 23700 cctccctatg agaggcgcag aaaaaaccat gattgctagt ggggaggtgc tccctctaca 23760 acccactcca taatctgccc ccgcccagct ctgaggccag ccccagggga aaatgccaga 23820 tccccaggga ggtgtgtgag acctcagggg ctccctcctc ccttacagca ggctcaggcc 23880 cctgggggcc tcagggccaa ggtctgtggg taagctacta tctctcactt gtcctctagc 23940 cacaaaagcc agggagatct ggcaatggac atgaggttct gaagaagcac atatgactgg 24000 cttcctaatg cgtggttgtt cagtgattca ataaacacgc atgggccagg catggggaaa 24060 tagacaaaca tgatccccaa cctctcccag agtgaactgg gagggaggag tgttcatccc 24120 tcaggattac accagagaaa caaaccagca ggagatatat atggttttgg ggggtcaaga 24180 aagaggaaaa acctggcaag gcaagtccaa aatcatagga caggctgtca ggaagggcag 24240 cctggaacct ctcaagcagg agctgatgct gcagtccaca ggcagaattt cttcttcctc 24300 ggggaaatct cagctttgtt cttaaggcct ttcaactgat tggctgaggt ctgccccttc 24360 ccccacattc tccaggataa tcttccttac ttaaagtcaa ctattaatca cagctacaaa 24420 atcccttcac agctacacat agatcagtgt ttgattgacg aacagcccct acagcctagc 24480 caagttgaca cataaaacta accatcacag ggggacaaat gatgtaaaca catcaacaaa 24540 taaaacagta acaagttaag gtctatggaa aaaacacaga aggggcagag agaaagaaag 24600 caagaaggag agtcccagtt tgctagggct tgtgggaagt ggggagcagt tctctttagc 24660 taggatattt gggaaaggca tatctgaagg agtgatattt gagcttagat taaaagatgg 24720 gaaggagcaa gccatgcaaa gagctaggat gttccaagca gagacggaac agcaagtgca 24780 aatgtcagga ggaatagaag gaggctggtg ggtggggtcc agtgagcaag aggagggcag 24840 gcaggagagg ggatggggag gtgggcaggc ccagaccacc cagggccctg gagactatcc 24900 tgatccaaca agggaagcct tgagtcactt cagtgtccat gtggagaatg gacctcagac 24960 tgaatgaggg aggcagtaag gagggcctct acctccaggg cttcgccctg tggactgcgc 25020 atagacatct ccaactcaga aagtctgaac eaaact'ttcc atagttcccc caagtctggg 25080 catcctccta ctcagtgaaa ggcagccatc acacctccct gccctgctcc cggatgcccc 25140 aaatcctctt ggtctccaag tccagaacct gagacttgtc cttgatgttt gtctttccct 25200 caccctttct gtattctggg aagatgggtt tttttccccc'agatgaatct gtaaaacttc 25260 tgtgatcaca ataaaaattc tggcagtatt attttctgga acatgacaaa gtgattcaaa 25320 attatttatc tggaagacta caaaacaaga atagccagga aatttctaaa aagaaagaag 25380 aaggaggagg agaaagaagg aggaggaaaa ggaggagaag aagaaaagaa aaagaaccaa 25440 gaaagggttc tagctctacc aaatattaaa acatatcatg aagctattta aaacaatatg 25500 gttgtggata ctgaaaaaga tgtgaataaa gtggaaggaa aataaataga aatgcacatg 25560 gggattgaga ctgtgaaaaa ggcagcatct cacatcagtg agggatgttc aacacctggt 25620 gttgggaaaa ctggctagtc atttaaacca aacaactggg tcctctacct cactcctgac 25680 attaagatac atttagatga ttcaaagagt aagacagaaa aaataacacg tgaaaacact 25740 atcagaaaac aacgtgggcc aggtgtggtg ggteaegeet gtaatcccag cactttggga 25800 ggccgaggca gacagatcac ctgaggtggg gagttcaaga ccagcctgac caacatggtg 25860 aaatcctgtc tctactaaaa atacaaaatt agctgagcgt ggtggcgcat gcctgtaatc 25920 ccagctactc aggaggccga ggcaggagaa tcacttgaac ctgggaggca gaggttgtgg 25980 tgagccgaga tcacgccatt gcactccagc etgggcaaca agagtgaaaa tccatctaaa 26040 aaaaaaaaaa aaagccaagg tggatatttt tatagtatca gggtagatca agcttctcca 26100 atcatgacat gaaacccaga aaccataaaa gaaaagaatg ataaaattgc ccacgtaaag 26160 taaaaagctt gcacacagaa aaacaccata caggttacaa gatgagcagc aaaatcagag 26220 aaaaaacatt gcaattcagg acacacagag gctattgttc ctaatattta aaaataaaag 26280 tagtggattg tctacaaaaa gatgaagaca agaatttcag aaaaccaaat actgcatgtt 26340 ttcacttaca agtggaagct aaacactgag tacacgtgta cacaaagaat ggaaccatag 26400 gccaggcacc gtggctcacg cctgtaatcc cagtactttg cgaggccgaa gcgggcggat 26460 cacctgaggt gaggagttcg agaccatcct ggccaacatg gtgaaaccca gtctctacta 26520 aaaatacaaa aattagccgg gcgtggtggt gggtgcctgt aatcccagct actcgggagg 26580 ctgcggcagt agaatcgctt gaaccctgga ggtggacctt gcagtgagcc gagatcgcac 26640 cactgcactc cagcctgggc aacagagtga gactccatct caaaaaaaaa aaaaaggaat 26700 agaacaatag acactggggc ctacttgagg gaggagggtg aggatcaaaa acctgcctat 26760 caggtactat gcttattacc tgggcggtga aataatctgt acaccaaacc ccagtgacat 26820 gcaatttacc gatgtaacaa acctgcccat gtacccgctg aacctaaaat aaaagttgga 26880 aaaaaatata gaaattttct ttgtaatagc caaaaactgc aaacagccca ggtgtctatt 26940 agtagaatgc ataaacaaac tcgggcatgt tcatacaatg taaaactact catcaataaa 27000 aagtgatact tctcagcaat gaaaagaaac tagctactga taccagctac aacatggatg 27060 gatttcaagt gctttatgat gagagcaaga agccagacac aaaagtgtct atatatatat 27120 acagtatata tacgtatata tacacatata tacagtatat atatacatat acatgtatat 27180 atatactgta tatatactgt atatatatac acagtatata tatacatata tacagtgtat 27240 atatactgtg tatatataca tgtatatata ctgtgtataVlitacatgtat atatactgtg 27300 tatatataca tgtatatata ctgtgtatat atacatgtat atatatgtat actgtatata 27360 tactgtatat atatatacac atatatacag tatatatata cagtatatac tgtatatata 27420 cagtatatac gtgtatatat acatatatac agtatatatg taaatataca tatatacagt 27480 atatatgtaa atatacatat atacatgtat atatatacac tatatatata catatatagt 27540 gtatatatac atatatacat gtatatattt actatatgat tccatttata taaagtgcca 27600 aaacagtcaa aaataatcta tgtggaaaaa atcaacaaag ggatcccccg ggctgcagga 27660 attcgatggc gcgccgacgt cgcatgctcc tctagactcg aggaattcgg taccccgggt 27720 tcgaaatcga taagcttgga tcggccgcaa taaaatatct ttattttcat tacatctgtg 27780 tgttggtttt ttgtgtgaat cgtaactaac atacgctctc catcaaaaca aaacgaaaca 27840 aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctcta 27900 tcgaggatct gcgatcgctg aattctgggg actttccact ggggactttc cactggggac 27960 tttccactgg ggactttcca ctggggactt tccactcctg cagcagtgga tattcccaga 28020 aaactttttg gatgcagttg gggatttcct ctttactgga tgtggacaat atcctcctat 28080 tattcacagg aagcaatccc tcctataaaa gggcctcaga ggaagtagtg ttcagctgtt 28140 cttggctgac ttcacatcaa agctcctata ctgacctgag acagagccat gaattccgtt 28200 tttttttttt tttttggaaa tatgagggtt tttccgcttc tgacagtgga acggaatgac 28260 agcagcacag gctggtgaat gactactttc tttataagca accaccttga gcctgaaatg 28320 gcagtcgcta gtctctattg ccttgctgtg gcctcgggat ggaaatctgc tggggaccct 28380 acagtcacct aatctctctc cttctcatcc ttctgtttca ttcagaggca gcctgccgcc 28440 cttctgggaa aagaccctgc aagatgcaag ccttcagaat ctgggatact aaccagaaga 28500 ccttttacct gagaaacaac cagctcattg ctgggtactt acaaggacca aatatcaaac 28560 tagaagaaaa gatagacatg gtgcctattg accttcatag tgtgttcttg ggcatccacg 28620 ggggcaagct gtgcctgtct tgtgccaagt ctggagatga tatcaagctc cagctggagg 28680 aagttaacat cactgatctg agcaagaaca aagaagaaga caagcgcttt accttcatcc 28740 gctctgagaa aggccccacc accagctttg agtcagctgc ctgtccagga tggttcctct 28800 gcacaacact agaggctgac cgtcctgtga gcctcaccaa cacaccggaa gagcccctta 28860 tagtcacgaa gttctacttc caggaagacc aatagtctag ctcgacatga taagatacat 28920 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 28980 ttgtgatgct attgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt 29040 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag 29100 ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tagatccatt 29160 taaatgttag atccggagag ctcccaacgc gttgggtcga cggcgcgcct accagtaaaa 29220 aagaaaacct attaaaaaaa caccactcga cacggcacca gctcaatcag tcacagtgta 29280 aaaaagggcc aagtgcagag cgagtaTata taggactaaia’liaatgacgta acggttaaag 29340 tccacaaaaa acacccagaa aaccgcacgc gaacctacgc ccagaaacga aagccaaaaa 29400 acccacaact tcctcaaatc gtcacttccg ttttcccacg ttacgtcact tcccatttta 29460 agaaaactac aattcccaac acatacaagt tactccgccc taaaacctac gtcacccgcc 29520 ccgttcccac gccccgcgcc acgtcacaaa ctccaccccc tcattatcat attggcttca 29580 atccaaaata aggtatatta ttgatgatgt tt 29612
<210> 3 <211 >29273 <212> DNA <213> Equine helper-dependent adenovirus-111-Ra <400>3 aaacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60 agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120 tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180 ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240 tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300 ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatatttgt ctagggccgc 360 ggggactttg accgtttacg tggagactcg cccaggtgtt tttctcaggt gttttccgcg 420 ttccgggtca aagttggcgt tttgatatca agcttatcga taccgtaaac aagtctttaa 480 ttcaagcaag actttaacaa gttaaaagga gcttatgggt aggaagtagt gttatgatgt 540 atgggcataa agggttttaa tgggatagtg aaaatgtcta taataatact taaatggctg 600 cccaatcacc tacaggattg atgtaaacat ggaaaaggtc aaaaacttgg gtcactaaaa 660 tagatgatta atggagagga tgaggttgat agttaaatgt agataagtgg tcttattctc 720 aataaaaatg tgaacataag gcgagtttct acaaagatgg acaggactca ttcatgaaac 780 agcaaaaact ggacatttgt tctaatcttt gaagagtatg aaaaattcct attttaaagg 840 taaaacagta actcacagga aataccaacc caacataaaa tcagaaacaa tagtctaaag 900 taataaaaat caaacgtttg cacgatcaaa ttatgaatga aattcactac taaaattcac 960 actgattttg tttcatccac agtgtcaatg ttgtgatgca tttcaattgt gtgacacagg 1020 cagactgtgg atcaaaagtg gtttctggtg cgacttactc tcttgagtat acctgcagtc 1080 ccctttctta agtgtgttaa aaaaaaaggg ggatttcttc aattcgccaa tactctagct 1140 ctccatgtgc tttctaggaa acaagtgtta acccacctta tttgtcaaac ctagctccaa 1200 aggacttttg actccccaca aaccgatgta gctcaagaga gggtatctgt caccagtatg 1260 tatagtgaaa aaagtatccc aagtcccaac agcaattcct aaaaggagtt tatttaaaaa 1320 accacacaca cctgtaaaat aagtatatat cctccaaggt gactagtttt aaaaaaacag 1380 tattggcttt gatgtaaagt actagtgaat atgttagaaa aatctcactg taaccaagtg 1440 aaatgaaagc aagtatggtt tgcagagatt caaagaaaat ataagaaaac ctactgttgc 1500 cactaaaaag aatcatatat taaatatact cacacaatag ctcttcagtc tgataaaatc 1560 tacagtcata ggaatggatc tatcactatt tctattcagt gctttgatgt aatccagcag 1620 gtcagcaaag aatttatagc cccccttgag cacacagagg gctacaatgt gatggcctcc 1680 catctccttc atcacatctc gagcaagacg ttcagtccta cagaaataaa atcaggaatt 1740 taatagaaag tttcatacat taaactttat aacaaacacc tcttagtcat taaacttcca 1800 caccaacctg ggcaatatag tgagacccca tgcctgcaaa aaaaaaaaaa ttagccaggc 1860 atggtagcat gtacctgtag tcccagctac ttgagaggtg aggtgggaaa atcactttag 1920 tgcaggatgt tgaggctgga gtgaactgtg attgtgccac tgcactccag cctggacaat 1980 agagcaagac cttgtctcaa aaaaatgcat taaaaatttt ttttaaatct tccacgtatc 2040 acatcctttg ccctcatgtt tcataaggta aaaaatttga taccttcaaa aaaaccaagc 2100 ataccactat cataattttt tttaaatgca aataaaaaca agataccatt ttcacctatc 2160 agactggcag gttctgatta aatgaaattt tctggataat atacaatatt aagagagact 2220 gtagaaactg ggccagtggc tcatgcctgt aatcccagca ctttgggagg ctgggtaaca 2280 tggcgaaccc tgtttctaca aaataaaaat attagctggg agtggtggcg cacacctata 2340 gtcccagcta ctcaggaggc tgaggtggaa ggatcgcttg aacccaggag gttgagactg 2400 cagtgaactg tgatcattct gctgcactgc accccagcct gggcaacaga gaccttgtct 2460 caaaaaaaaa aaaaaaagag acaaattgtg aagagaaagg tactctcata taacatcagg 2520 agtataaaat gattcaactt cttagaggaa aatttggcaa taccaaaata ttcaataaac 2580 tctttcccct tgacccagaa attccacttg aataaagctg aacaagtacc aaacatgtaa 2640 aagaatgttt cttctagtac agtcggtaag aacaaaatag tgtctatcaa tagtggactg 2700 gttaaatcag ttatggtatc tccataagac agaatgctat gcaaccttta aaatatatta 2760 gatagctcta gacacactaa tattaaaagt gtccaataac atttaaaact atactcatac 2820 gttaaaatat aaatgtatat atgtactttt gcatatagta tacatgcata ggccagtgct 2880 tgagaagaaa tgtgtacaga aggctgaaag gagagaactt tagtcttctt gtttatggcc 2940 tccatagtta gaatatttta taacacaaat attttgatat tataatttta aaataaaaac 3000 acagaatagc cagacataca atgcaagcat tcaataccag gtaaggtttt tcactgtaat 3060 tgacttaaca gaaaattttc aagctagatg tgcataataa taaaaatctg accttgcctt 3120 catgtgattc agccccagtc cattaccctg tttaggactg agaaatgcaa gactctggct 3180 agagttcctt cttccatctc ccttcaatgt ttactttgtt ctggtcccta cagagtccca 3240 ctataccaca actgatacta agtaattagt aaggccctcc tcttttattt ttaataaaga 3300 agattttaga aagcatcagt tatttaataa gttggcctag tttatgttca aatagcaagt 3360 actcagaaca gctgctgatg tttgaaatta acacaagaaa aagtaaaaaa cctcatttta 3420 agatcttact tacctgtcca taattagtcc atgaggaata aacacccttt ccaaatcctc 3480 agcataatga ttaggtatgc aaaataaatc aaggtcataa cctggttcat catcactaat 3540 ctgaaaaaga aatatagctg tttcaatgag agcattacag gatacaaaca tttgattgga 3600 ttaagatgtt aaaaaataac cttagtctat cagagaaatt taggtgtaag atgatattag 3660 taactgttaa ctttgtaggt atgataatga attatgtaag aaaacaacag gccgggcggg 3720 ttggttcaca cgtgtaatcc cagcactttg ggaggctgag gcaggcagac tgcctgagct 3780 caggagttcg agaccagcct gggcaacacg gtgaaatccc gtctctacta aaaatacaaa 3840 aaaattagcc gggtgtggtg acacatgcct gtagtcccag ctacttggga ggctgaggca 3900 ggagaatcac ttgaacctgg gaggtgaagg ttgcagtgag ccaagatggc accacttcac 3960 tccagcctgg gaaacagagc aagactctgt ctctgagctg agatggcacc acttcactcc 4020 agcctgggaa acagagcaag actctgtctc aaaaaaaaca aaacacacaa acaaaaaaac 4080 aggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggcgggtgg 4140 atcacctgag gtcaggagtt ccagaccagc cttgtcaaca tggtgaaacc tccccccgcc 4200 gtctctacta aaaatacaaa aattagccag gcgtggtggc aggagcctgt aatcccagct 4260 acttgggagg ctgaggcagg agaatcgctt gtacccagaa ggcagaggtt gcactgagct 4320 gagatggcac cattgcactc cagcctgggg gacaagagcg agatttcgtc tttaaaaaac 4380 aaaaacaaaa caaaaaacca tgtaactata tgtcttagtc atcttagtca agaatgtaga 4440 agtaaagtga taagatatgg aatttccttt aggtcacaaa gagaaaaaga aaaattttaa 4500 agagctaaga caaacgcagc aaaatcttta tatttaataa tattctaaac atgggtgatg 4560 aacatacggg tattcattat actattctct ccacttttga gtatgtttga aaatttagta 4620 aaacaagttt taacacactg tagtctaaca agataaaata tcacactgaa caggaaaaac 4680 tggcatggtg tggtggctca cacttgtaat cccagtgctt tgggaggctg agacaggaga 4740 gttgcttgag gccaggagtt caagaccgac atggggaatg tagcaagacc ccgtccctac 4800 aaaaaacttt gtaaaaattt gccaggtatg gtggtgcata cctgtagtcc cagctactcg 4860 ggaggcggag gcagaaggaa tcacttgagc ccaggagttt gaggctgcag tgagctacga 4920 tcataccaca gcactccagc gtggacaaca gagtaagacc ctatctcaaa aacaaaacaa 4980 aacaaaacaa acaaaaaaaa ccacaagaaa aactgctggc tgatgcagcg gctcatgcct 5040 gtaatcccag tattttggga ggcccaggtg ggcgtatcac ctgaggtcag gagttagaga 5100 ccagcctggc caacatggtg aaaccccatc tctactaaaa atacaaaatt agccaggcat 5160 gtggcacgcg cctgtagtcc cagttactgg gaggctgaag caggaggatc acctgagccc 5220 gggaggtgga ggttgcagtg agccgagatc acaccactgc actccagcct gggtgacaca 5280 gcaataccct acctcaaaat aaaaaagaaa aagaaaagaa aagttgctgt ccccgctacc 5340 ccaatcccaa atccaaacag cctctctcat ctcacagtaa gggggaaaaa tcacccaaaa 5400 aagctaagtg atcttttgaa aacccaaact cttagaagtc taagattatt atagtcaact 5460 catgaagtgt catcataaaa gatactctaa tattatttaa gtagaaccac atattggttg 5520 tcttggtatg tctagcccct ggcatacaaa atatttaata acactgatat ggtacctgtg 5580 atgtgaaaat gtactatgag tacagcttta taaatactat atatgtacct atatacagaa 5640 aaaaatacaa caaaatcata aaagcactta tctttgaaag aggagttaca gcaattttat 5700 ttagttcttt attgctttgc tatatattct aaattttttt caatgaatat atatcacttt 5760 taaaaaaatt caatggtctt tcttataaat tatctttggc agcatgcgtt tttatatata 5820 catataaaat gtatgggaaa tttttaaagg atacattaaa ttaaagcaaa atatacaaac 5880 aaaaaatcag aatacaaaaa gataaaaaga ttgggaaggg agggagggag taaggaggaa 5940 gggtgggtgg gtatagagaa atataccaaa taatggtaag aagtggggtc ttgacacttt 6000 ctacactttt tttaaataaa aaaaattttt ttctctctct tttttttttt tagagacgaa 6060 gtctcgctat gttgcccagg ctggtcttga actcctggga tcaagagatc ctcctgcctc 6120 agcctcccaa ggtgcttgga ttacaggtgt gagccaccac gcctggtcac tttctacact 6180 ttaatatata tattttttca ttttcaatgt catttttatt agttaattta taatacccat 6240 tcaccattat attcaaagtc tatttgaaga aataaaccag aaagaatgaa atactctagc 6300 tcacatgcta ttcaatacta aattaccttt caaatcacat tcaagaagct gatgatttaa 6360 gctttggcgg tttccaataa atattggtca aaccataatt aaatctcaat atatcagtta 6420 gtacctattg agcatctcct tttacaacct aagcattgta ttaggtgctt aaatacaagc 6480 agcttgactt ttaatacatt taaaaataca tatttaagac ttaaaatctt atttatggaa 6540 ttcagttata ttttgaggtt tccagtgctg agaaatttga ggtttgtgct gtctttcagt 6600 ccccaaagct cagttctgag ttctcagact ttggtggaac ttcatgtatt gtcaggttgg 6660 cccgtaatac ctgtgggaca acttcagccc ctgtgcacat ggccaggagg ctggttgcaa 6720 acattttcag gtaggtggac caggacatgc ccctggtcat ggccaggtgg aggcatagtg 6780 ctatacagca ggcagaagtc aatattgatt tgtttttaaa gaaacatgta ctactttcat 6840 aagcagaaaa aatttctatt cttgggggaa aagattatgc cagatcctct aggattaaat 6900 gctgatgcat ctgctaaacc ttcacatatc agaacatatt tactatagaa agaatgaaaa 6960 tgggacattt gtgtgtcacc tatgtgaaca ttccaaaaat attttacaac aactaagtat 7020 tttataaatt ttatgaactg aaatttagtt caagttctag gaaaatacaa accttgctag 7080 atattataaa aatgatacaa tatatattca tttcaggctc atcagaatat atctgttatc 7140 acctgacaag aatgaaaatg caccattttg tagtgcttta aaatcaggaa gatccagagt 7200 actaaaaatg acttcttcct tgaagcttac tcaccaactt cctcccagtt actcactgct 7260 tctgccacaa gcataaacta ggacccagcc agaactccct tgaaatatac acttgcaacg 7320 attactgcat ctatcaaaat ggttcagtgc ctggctacag gttctgcaga tcgactaaga 7380 atttgaaaag tcttgtttat ttcaaaggaa gcccatgtga attctgccca gagttcatcc 7440 cagatatgca gtctaagaat acagacagat cagcagagat gtattctaaa acaggaattc 7500 tggcaatata acaaattgat ttccaatcaa aacagattta cataccatac ttatgtcaag 7560 aagttgtttt gttttattgc atcctagatt ttattttttt gatttatggt ttactttaag 7620 cataaaaaat ttgtcaatac aactcttccc aaaaggcata aacaaaaatt cataaaactt 7680 gcatcacttg agatacttca ggtatgaatt cacaactttg ttacaactta ctatatatat 7740 gcacacatat atatatattt gggtatattg ggggggttct aatttaagaa atgcataatt 7800 ggctatagac agacagttgt cagaacttgg caatgggtac gtgcaggttc attataccaa 7860 gtctacttgt agttgttcaa aatgtatcat aatacaaggc cgggcgaggt cgtcacgcct 7920 gtaatcccag cattttggga ggctaaggca ggaggattgc ttgaggtcag gagtttgtga 7980 ccagcctggg caacagagca agaccctgtc tccaaaaaga aaaaaaataa ttttttacaa 8040 aataaaaaca aaatgtatca tcagacgaaa ttaaataaga ggcaattcat ttaaatgaca 8100 acttttccca gcttgacatt taacaaaaag tctaagtcct cttaattcat atttaatgat 8160 caaatatcaa atactaattt tttttttttt tttttttttg agacggagtc tcgctctgtc 8220 gcccaggctg gagtgcagtg gcgcgatcct ggctcactgc aagctccgcc tcccgggttc 8280 acgccattct cctgcctcag cctcccgagt agctgggatt acagacatgc gccaccacgc 8340 ccggctaatt ttgtattttt agtagagatg gggtttctcc atgttggtca ggctggtctt 8400 gaatttccca cctcaggtga tctgcctgcc tcagcctcac aaagcagtag ctgggactac 8460 aggcacccac caccacactt ggttaattct tttgtatttt ttttgtaaag acgggatttc 8520 accatgttag ccaggatggt ctcgatctcc tgatctcatg atccgcccgc ctcagcctcc 8580 caaagtgctg ggattacagg cgtgagccac cccgcccggc catcaaatac taattcttaa 8640 atggtaagga cccactattc agaacctgta tccttatcac taatatgcaa atatttattg 8700 aatacttact atgtcatgca tactagagag agttagataa atttgataca gctaccctca 8760 cagaacttac agtgtaatag atggcatgac atgtacatga gtaactgtga acagtgttaa 8820 attgctattt aaaaaaaaag acggctgggc gctgtggctc atgcctgtaa tcccagcact 8880 ttgggaggcc aaggcaagtt gatcgctcga ggtcaagagt tcgagaccag cctggccaac 8940 gtggtaaaac cccgtctcta ctaaaaatac aaaaaaaaaa ttagccaggc atggtggcac 9000 aggcctgtaa tcccagctac tagggaggct gagacatgga gaactgcttg aatccaggag 9060 gcagaggtta cagtgagccg agatcatacc actacactcc agcctgagtg acagagcgag 9120 actcctgtct aaaaaaaaaa aaaaaaaaaa agatacaggt taagtgttat ggtagttgaa 9180 gagagaactc aaactctgtc tcagaagcct cacttgcatg tggaccactg atatgaaata 9240 atataaatag gtataattca ataaatagga acttcagttt taatcatccc aaacaccaaa 9300 acttcctatc aaacaggtcc aataaactca atctctataa gagctagaca gaaatctact 9360 tggtggccta taatcttatt agcccttact tgtcccatct gatattaatt aaccccatct 9420 aatatggatt agttaacaat ccagtggctg ctttgacagg aacagttgga gagagttggg 9480 gattgcaaca tattcaatta tacaaaaatg cattcagcat ctaccttgat taaggcagtg 9540 tgcaacagaa tttgcaggag agtaaaagaa tgattataaa tttacaaccc ttaaagagct 9600 atagctgggc gtggtggctc atgcctgtaa atcccagcac tttgggaggc tgaggcgggt 9660 ggatcacctg aggccagaag ttcaagacca gcctagccaa catggcgaaa ccctgtctct 9720 acaaaaaata caaaaattag ccgggtgtgg tggcacgtgc ctgtagtccc agttacttgg 9780 gaggccgagg caggagaatc gcttgaacct aggaggtgga ggctgcagtg agccgagatt 9840 gtgccactgc actccacttc agcctgggcg acaagagcaa gactccgtca caaaaaaaaa 9900 aaaaaaaaaa aagcttaaaa tctagtggga aaggcatata tacatacaac taactgtata 9960 gcataataaa gctcataatc tgtaacaaaa tctaattcga caagcccaga aacttgtgat 10020 ttaccaaaaa cagttatata tacacaaaaa gtaaacctag aacccaaagt tacccagcac 10080 caatgattct ctccctaagc agtatcaagt ttaaagcagt gattacattc tactgcctag 10140 attgtaaact gagtaaagga gaccagcacc tttctgctac tgaactagca cagccgtgta 10200 aaccaacaag gcaatggcag tgcccaactt tctgtatgaa tataagttac atctgtttta 10260 ttatttgtga cttggtgttg catgtggtta ttatcaacac cttctgaaag aacaactacc 10320 tgctcaggct gccataacaa aataccacag actgagtgac ttaacagaaa cttatttctc 10380 acagttttgg aggctgggaa gtccaaaatt aaggtacctg caaggtaggt ttcaatctca 10440 ggcctcttct ttggcttgaa ggtcttctaa ctgtgtgctc acatgacctc ttctaacaag 10500 ctctctggtg tctctttttt tttttttttc ttttttgaga cagagtctca ctctgtcacc 10560 caggctggag tacagtggca caatctgggc tcactgcaac ctccaactcc cgggttcaag 10620 tgattctcat gcctcaccct cccgagtagc ttggatgaca ggagcccgct accacaccca 10680 gctaattttt gtatttttag tagagatggt gtttcactac attggccagg ctggtctcaa 10740 actcctgacc tcgtgatcca cccaccttgg cctcccaaag tgctgggatt acaggtgtga 10800 gccactgcgc ccgtcctggt gtcttttcat ataagggcac taatccaatc agacctgggc 10860 ccaaccctcc cgacttcttc taactgtaat taccttccaa aggccctgtc tccaaatacc 10920 atcacactgg gggttaggac ttcaaaaaag gtatgggggg ggtgtgggag gacataaatg 10980 ctcagtccat aacaagcacc caacataaaa atggctagaa cagatcacaa aaaaaaggtc 11040 ctgtatggct ttggggaagg gctcaacccc aaaatatctg agagctctgg aggggcctag 11100 aagtggtaaa tgaatgaaaa cgtggttact ctccagatct gcctttccca aatatggcca 11160 ttcttggctg aatcagaaat caaaggacag gttattaatt actagctcta agttacttac 11220 catttgctga gacagttcag aaatctgact gcatctcctc agagatctag aacacagttc 11280 tcaaattcta acttacttgt gatatacttg tgaatgataa aaatcgctac aggtactttt 11340 attaatctga aagagtattg agaaattacc tttcattctg acttttgtct ggaatgaaaa 11400 tcaatacttt tgctataatc gattactgaa ataattttac tttccagtaa aactggcatt 11460 ataatttttt ttaattttta aaacttcata attttttgcc agactgaccc atgtaaacat 11520 acaaattact aataattatg cacgtcacat ctgtaataat ggccttcatg taaacatttt 11580 tgtggtttac acataaaatc tctaattaca aagctatatt atctaaaatt acagtaagca 11640 agaaaattaa tccaagctaa gacaatactt gcaacatcaa ttcatcatct gtgacaagga 11700 ctgcttaagt ctctttgtgg ttaaaaagga aaaaaaaaaa aaagacatgt tggccagatg 11760 cggtggctca cacctgtaat cccagcactt tgggaggctg aggtgggcgg atcacccctg 11820 gcctgcccaa catggtgaaa ccccgtctct actaaaaaca caaaaattag ctgggcgtgg 11880 tggcgggcgc ctgtaattcc agctactcgg gaggctgagg caggagaatt gctagaaccc 11940 aggaggcaga gattgcagtg agctgagatt gcaccattgc actacagtct gggcaacaaa 12000 agtgaaactc catcttaaaa aaaaaaagac aatgttcgtg ggtccaaaca agacttaatg 12060 gaagtgagtc taaaaatgag ctatgtgggc caggcgtagt ggctcccacc tgtaatccca 12120 gcactttggg aggccgaagc aggcagatca tgaggtcagg agatggagac catcctggcc 12180 aacacggtga aatcctgtct ctacaaaaat tagctgggcg tggtggtgcc tgcctgtaat 12240 cccagctact cagaaggctc aggcaggaga atcgcttgaa ccagggagtc ggtggctaga 12300 gtgagccgag atttgcatca ctgcactcct gcctggtgac agagcaagac tccatctcaa 12360 aaaaaacaaa caaaaataaa agataaaaat gagctatgtg aattaaaaga ggtataacaa 12420 tagataaacc atattttatt taattcctag taatgagtaa tatttccaaa cttctggaat 12480 gggcagaaat tgctagttgg catattttta ccttttatat tcagatacat taaaattctc 12540 aaaaaaaaac acctcaaagc agatgatccg ccatctcctt ggataatttg tgttaactca 12600 ggataacaga aaaccaaaat tatgagttac tgatgcaata ttcctaaatg taaaaataat 12660 taaagctaat agtagattca tcttccaatt tcatatcagt cttacaaata aactacatat 12720 ataacttgct tgccttccct tctgagggat aaagctgtta gaagaattaa aatcagcatt 12780 cttgactatt caaccaaggg agggataaat tattactcat tctagggaca tgggctcata 12840 actactacat gtgtaaggac atgaatttac ccaatattac aatttttcct tttattagtg 12900 tgtacagtgg aagaatagac atgttcactc tggacaaaaa aaaaattata cttatcagtt 12960 atcagaagca caatgctgaa gacagtagtt ccataacaat ttgaagtatg tgatcgaact 13020 agtagattat cttagtagta gtgaattatt gtaaatgtta gtaatttggc agccactggg 13080 cagaaaaata agaattgagg ctcaatattg atattaatgg tggtgattga cacataaatt 13140 ttatcaagtc tacacaatat aaaattacag aaaggtagaa gagtatacca gtacaacttc 13200 aacatatctt cactacaagg gagtaaaatg acatggccta gttactatct aatgaactgc 13260 agaaaactaa aagaaaactc caaggcaact cttctctgct gatctggttg gtccttttcc 13320 taccttrtgc aatacccaga tacaaacaat ggatagaaaa caaagtagac ttgtagtatg 13380 caggtcacag tgctaaattc acagaaagaa acccctgaac tgaactgctc tatttcctgg 13440 tggtcacaaa gagtaattct ggtttacacc tacagattga tgtcaatcta caccctgttg 13500 ataacagtgt ggccaaggac aaaaaaaagg tgctccgttt taccaattct gtaaaaaatt 13560 attggcaggg taagctcggc tagggcagga ttacatttct aggactacca tccccgaaat 13620 ttagaagata ttatatccac ataaagcata tctttcacat taatttgcaa aaatctaaaa 13680 gctttttctt agctcaagtg tgtccaagtt taccctggca gtttaaaacg atagttacaa 13740 gcagcatggg ttgtatcaga cacatttgag ggccaatttc atgtaagtga tattgggcaa 13800 gttacttcaa ctatctgtgc ctccaaggtc atactagtgt ttatttacct aaagggtacc 13860 tgttatgtaa ctttagggtg tttacattag ataatgcctg caaaatattt acttcaacgc 13920 ctaaaacata gttaagtatt caataaatac ctactattgt cactactaac ttaaaagttt 13980 agagattaag agcagaatct ggggtgagac aaacttaggt tcaaatccta gtattgttgg 14040 gtaatcttgg gcaagttact taacctctct gatttgtgta atttaaaaaa ttagttaata 14100 tacataacag ggcttagaag agtatctagc acatagcacc atttaagcat ttgttattgc 14160 taacatgcaa acaatttaag ggaaagaaat tttttaaaaa ggaagaggga tttgcaaact 14220 aaaaacaatg agtatcttat gttcaaagaa aactaacaaa cagccagctc tagcaataat 14280 taaattcact atatactggg gcaggcatca caccccaaag ctaaaagcgt ctacctaggc 14340 caggcacggt ggctcatgcc tgtaatccca gcactttggg aagcagaggc gggcagatcg 14400 cttgagctca ggagttcaag accagcctgg acaacatggc aaaacaccat ctctacaaaa 14460 aatacaaata ttaggccggg cgcagtggct cacgcctgta atcccagcac tttgggaggc 14520 caaggcgggt ggatcacctg agatcaggag ttcgagagta gcctggccaa catggtgaaa 14580 cctcgtctct attaaaaata caaaaaatta gccaggcatg gtggcaggcg cctgtaatcc 14640 cagctactca gggggatgag gtaggagaat cgcttgaacc cgggaggcag aggttgcact 14700 gagccgagat catgccactg tactccagcc cgggcaacaa gagcgaaact ccatctcaaa 14760 aaataaataa ataaataaat aaaataaagt acaaatatta gccagggatg gtggtgcgca 14820 cctgtagtcc cagctacttg ggaggctgaa gtgggagaat cccctgagcc tggggagaat 14880 cacccgagcc cgggaagtcg aggctgcagt gagcagtgat tgtgccactg cactccatcc 14940 taggtgacag agtgagaccc tgtctcaaaa aaaagaaatt ggcagaatta agtaagttga 15000 tgtttagaga tgaaaaatca acattttttc ctcagcaact gaataaaaac aacagccact 15060 accatttttt tgagtaccta tttgtagcct attttttaac tggtattact cgagagagag 15120 agagctaggt tcgagacaga gctccttctc ttaataactg tatgacctag ggtatgtctg 15180 ttagcctctc tgaggcttca aaggttcctc atctgtaaaa tggtaataat cataccattg 15240 ctacagggct gttttgaaga ctaattagga ctatgtaagt aaacatgatg atggctatta 15300 ttactgttcc ccgccagggg ccatgcaagg gttgctgatt cacatagact gtcttataat 15360 cctctcaata actccaagag gtagccagca cctcagatat acataaaatg acttaagccc 15420 agagaggtga agtaagttgc ccacagccac acaactagta aatagcccaa acaagctgga 15480 ttcccagtta gactccgtta atagcactgc tctttacctt aagtcattac aatgcctaat 15540 atgaaataga atcgcttctt tcttagggtt caagtggtta attatttaat gtattcattc 15600 aacaaaccat catcgaggac ctcttacaag ccaagtactg tgctaagtgc tagagttacg 15660 gcggtgattc ctgcccttaa aaagttttag tgggagaaac aacaggtaac caggtcattg 15720 ccaaaacaac aaaaataatc ataataaagc aggctaaagc atatttaact ggccggggtt 15780 ttgactattt tagcaagcat gatcagaacg gttgaggagg gaggccagca gcttggccgg 15840 ttcaacaaac aagaaaaaac cagtgagggt ggagctaaga taccagaggc tgattacggt 15900 taagaatgtt cttgaaggta aggaccagat tctcattttc tatatcctgg ggcatcggtc 15960 agcatggaat ctggattcta gcacatgtga atttcggctt gaaatgacct aatgcctttt 16020 ccctagttcc ttcgtgtgtc aaatacgcat ggttaccgct accagagctg tagtggggct 16080 tcaatgaggc catgagcatc tccataaaga tgaactacag tgtgtgcaaa actaaaggca 16140 aaacctggtc cccacacgcc ctcccaggtg gtcgctttcc gtgccgaggc ccctccagag 16200 gtgccccgag aacctcacca tcgcacccca aacttccagg gaagggcctc tcccgagaaa 16260 gcccccacgc ccccaccccg cgccatcatt cccgaatctg ccctcggccc ctccccgcag 16320 cacgctcgca ggcggcacat gtcaaccaaa acgccatttc caccttctct tcccacacgc 16380 agtcctcttt tcccagggct cccccgagga gggacccacc ccaaaccccg ccattccgtc 16440 ctccctgccg ccctcgcgtg acgtaaagcc gaacccggga aactggccgc ccccgcctgc 16500 ggggttccct gggcccggcc gctctagaac tagtggatcc caattgaagg cctggtctaa 16560 atgactccaa aatcaccact taattcaaga gactgatttc cctgagtcag gccccttaaa 16620 gcagctattt caatgggaca gggaaacaac cctaggatct ggattagaat cacttggggg 16680 ctgccacacc cccagggctc tgatcctgcc cttctcccac acgcacattc acatactgct 16740 gcagtgacct tccatttcta atgggttcct gggccatctg tcaggtatag ggaatggaaa 16800 aggggttggg gaggctctgc ttcagaaagt ttgtgtcagg ggctcccaga gcctccacag 16860 atagatagca ggggtcccca ccctaccatg gcagctataa atgtgatcaa catttattgg 16920 cctaggatac agcagttagc aaaatgcctg atgtagttcc cactccgtgg aggttgcagg 16980 ctagccaaga agtcatgagt tcagcaaccc ttacgcacca gtgggatgag attggaccag 17040 gccgagggta gtcttgggaa cactcagcat ttgtctgagg gccagaagag gctgcttgcc 17100 ctcagacagg aggtcagcat ctttattgta gcccatgaca cctctacacc attgctcttc 17160 tggtcttatg gaagacatct ttgggcctga taacagcgga gtctgtgtcc cacttgtcca 17220 ggctggagtg ccacatcagg cacactccag ttgcagggac agcacagaca agtttcagga 17280 aggctggtgg cctccaggag gttaacctta taaggccaga ttgtaaccta gttgaaaaac 17340 atacacatgc catgataata aaagaaccta ggcaccatta caagagaaaa aatcattttt 17400 gtagatacga gcatggattc ttgggtgggt cagacacact gggcttgtgc tctgactgca 17460 ctgtctcccc tacctgacct tgggtaaacc ataagactgc tgcatgactc agtgtccacc 17520 ccaaaaaagt accggtagat attggccaca gtagatatca gctagagtgg actctcatga 17580 caatgagggg agatgtattc cccatcttag gcacctggga ctctaccttc catcttctgc 17640 tccgtgtctc tccatcccca ggctcttcag aactcaggga gtccagaatg tcagctccca 17700 gatttcagcc ttcagaaagg aaacccatta ccgttcagtt gaacaaatgt tgtctgagcc 17760 ccagatctgg gctcagaggc catctaggct atgagacaag aggggaacaa agcaccgtct 17820 gcactcactc accacactca cttgctgtcc caggtcacat ccatcgggta gagaatctaa 17880 gaggctgagc tagctcccgc caccagccca gcccacccca cctggcccct tccttccttc 17940 tacaaaatat gcaccacctg tcaaagggtg ggcagtgcca ggcctgcata cagagcactg 18000 agtgtaaaag cagacatgga ccctgacctc caggagcttc caattttctt gaagagacaa 18060 atcagctggc atttcagtcc agtgtgatct gctcttggtg agcacagacc tagggagttg 18120 gggcagcttc ccagaagaac tgcagtccag gctgagggca gagaaatgag gggaatggcg 18180 aggaattggg gagcaggggg gagctcagta gagagccaag ggcgggaggt gagaagtccg 18240 tgttgggcca ggagctaccc tccggtggcc acagccgaag tcgaggatgc ctttggaact 18300 catccccact tctctctttc tgtatgtagc cgtccaagaa caagtcacct ccaagtgtag 18360 ccggatcaag gcaagccccc catctagcaa gcacttgatg ccacccagaa ctgggcttct 18420 tcagaacaat ctgagtccag gaatgatccc actcaccagg caccagagct gcgagggcat 18480 gggagtgatc tcaccaactc tggggaagcg gcaaggaatt ttcacctcca gcccccagtg 18S40 tcccatcctc tcacactcag gccagactcc cctgggcaga cttgactctg tctgccagca 18600 tatgcagagc cccaaggcca ccccaccaga agtgcccctg cctgggttct gtcccagctc 18660 cctgggcacc cagtccttga gtccccacca gctcagacgg cctagtgtgc caagaatgcc 18720 cactgcgttc aacaatgctg catgggtcac agcggcagca gctgtgacca cagcagtttc 18780 ggggaaaaca cccctcagcc aagtggataa cagcgttcag cagcactcac cttctggcca 18840 ggcctgcctt cagaggccat ctgattggga ggcacaagtg cccgctgcga tgggaacaca 18900 agtgcccctg gccaacaacc ccagcttcag cctgctgggc agccagagcc tcaggcagag 18960 cccggtacag ggcccggtgc ctgtagcaaa caccaccaag ttcctccagc agggtatggc 19020 cagctttagt cccctgagcc ccatacaggg catcgagcca ccaagctatg tggctgctgc 19080 tgccaccgct gctgctgctt ctgccgttgc tgccagccag ttcccaggtc cgttcgacag 19140 aacggatatt ccccctgagc tgccacctgc cgactttttg cgccagcccc aacccccact 19200 aaatgatctg atttcgtcac ctgactgcaa tgaggtagat ttcattgaag ctctcttgaa 19260 aggctcctgt gtgagcccag atgaagactg ggtgtgcaac ttgaggctga tcgacgacat 19320 tttggaacag catgctgctg ctcaaaatgc cacagcccag aattctgggc aagtcaccca 19380 ggatgctggg gcactttaaa tctgagcagg atgcccatag aaacccccat ggtgacatca 19440 ctctaggaag tggtgtcgat ccatacccgc agttgtctcc cgttacaatt tgagtggtgt 19S00 tgtcagccca tgcttatccc tctctctacc tgtgacaaaa tggaaagctg gtgatttttc 19560 aagctacgtg tacatatttg aaaattttgt aaatggtttt cctaaacatt aatgacagaa 19620 gtatttatac ttcattttgt gactttgtaa ataaagcgac ggcttttgtt tcagtagagt 19680 tgtgtttact atgcattgtt ttgtgtttat tatacaatgt tacaaatatg cagaccgtgt 19740 tgtttgctcc agtgatacct tgttaagcta ggtggctgag tcgcttatgg ttttaatgca 19800 atgagcaatg tggatatgac caagagttgt tgtgcaagtt gacaaatgcc aaatagaaaa 19860 ccacttggcc atttatttct atgttcacta aaaatcctat tgccttgtgt gattcttaat 19920 ctcttttgcg aacctttcag tctccgctag ctctttccta atgagcttta cagcagaagc 19980 tgttttatcg ttaagtgccc cacagagaca ctttaccagg aggctgggag agttctccag 20040 atttgggaga ggcgcagaga cagtgtgtga gccgagccct gtctcagcaa tccacctgga 20100 ggagctagag tatcctcctc cctttaccat tcagaccgag agaaaaagcc cagcttgtgt 20160 gcaccctcgt ggggttaagg cgagctgttc ctggtttaaa gcctttcagt atttgttttg 20220 atgtaaggct ctgtggtttg ggggggaaca tctgtaaaca ttattagttg atttggggtt 20280 tgtctttgat ggtttctatc tgcaattatc gtcatgtata tttaagtgtc tgttatagaa 20340 aacccacacc cactgtcctg taaacttttc tcagtgtcca gactttctgt aatcacattt 20400 taattgccac ctcgtatttc acctctacat ttgaaatctg gcgtctgttt eaagccagtg 20460 tgttttttct tcgttctgta ataaacagcc aggagaaaag tgcctctatg tttttatttt 20520 tcaagggagt attcagtacc tacaaaccca agtcaggaag cctgctagtg gctttggttc 20580 tttcagaggc tgctcgatgc cttgtgtgtc agaaagaaag attcagcagt tttgcatcat 20640 ggcaaagaag cctgttattt tggggctcag cccctcattt tatagaggat gaaacagagg 20700 gggatgggag gtcacaaaga caactgcccc gggagcaggt gtgggggaga cttgccctga 20760 gggtctagac gctctgcacc accgtcctgt ctcccttgct gaagaccaca catgcccttc 20820 tttgaccaga ccctgccacc tgataggcca ggacctggta ggcgggtacc caggtttcat 20880 ggatggaacc acatctcccc aaaagtgggg aggtagctac tgggatgcac gcctcccgcc 20940 atgtgctata ggagagcagc tgaagcaaca gttgggatca gatgtagtca caattgaatg 21000 catcatcaca tttatccctc taagtggctg ggagagttga tatcctcatc cctaaggtac 21060 aaaatgttcc aatttgatca gtggctttca ggagctgaga aaggcatgtg ctctgaggca 21120 gagctgttat gtcccgcaga gcctaaaaat gctctaagaa catgctccct gccaaaattc 21180 tcaatggctg tgacaaggga caacgatcga ccaatggggg tggaagcaga cctccgcagt 21240 ccaggggcca gagctaggac agaggggtcg gagaaagagt cattttccca acactccagc 21300 tcttggccag tcctcacaca gtcccctcct gcttcctgct gagagagata tcctcatagg 21360 tctgggtaaa gtccttcagt cagctttcat tccctgtcac caactttgtc tctgttctcc 21420 ctgcccgtct caggcagcac tcctcaggaa acctctccaa gagccagcct cactgcagcg 21480 cccactattg tccctctgcc tcaagtgtcc catccatgcc aggccccagg caggctgcag 21540 ctttccctca gggccacacc aaagcacttg ggctcagctg tgctgtcccc ctccatcact 21600 gagctcaggg gcagcagggg tggggtgcca ggaggcccat tcacccttct ctggctctgt 21660 gttggaccca cctgcccagc cactgctgct tagaacctac ccgctgggaa aatgaagccc 21720 tcccggaggg gccacctcaa cctgagagcc tcacggatca cagttgtccc cactcagctc 21780 tgccagccct cagagaccca tagataaaag ctgagcttgg ctcgcagagc tggttccatc 21840 ttccattccc agagggttca acttcctacc ccaaccacac agggaacctc aaggctgagc 21900 cagtgtgggc tgcagtgcag accagcttcc tggacacgtc ctgccacctg accccaggct 21960 ggcctcactg cccctggcac tcctgaccct atcctcattc ctcctggcag tgcgtgttct 22020 gccattccgc tttcccttag ctgtcctctc actgtactgt cagcttctcc ttttccaggt 22080 gccccccagg ggctttccac atgaccctgt caccccacag cccatccagc accaattcca 22140 gctctctgcc acccttcaaa ggagtgacag tgccctgctt cacctcccac tcacccctca 22200 acccagagca atctggctcc agtcttgcct ccttccccct aagtactcta gtcacagttc 22260 caaattcctc ctggtcataa agccaaatga agcttcctgg tcctcagcgg acttgccact 22320 tcagcagtac tggactctct cctcccagaa acctgtttcc ccttggctcc tggagcccac 22380 actctgctgg aatccttctg cctctctggc ctgtagcctg gccctctctc ccaacctgag 22440 gtceattctc tcctgctcct ccacaagatg ttgctccttc cattacttcc tccctctcaa 22500 ccaaagctcc ttcattagct ctttatcttc tggtttcttc ccctgggcag acgaatggat 22560 tcaagagcct gtggcccagc agcccagcac tccaggatct cagcacttca gcatcccagt 22620 accctagcat ctcaataccc cagcacccca gcaccatagt attccagcac cccattgtcc 22680 aagcatctca gcactccagc atcccagcac cccaacactc cagcagccca gaatctcagc 22740 accctagcac tgcagcatct caggacccca gcacttcagc atcccagcac actagtactc 22800 cagcatctcg gcaccccagc acctaggcat cccaacaccc agcaccccag cacttaagca 22860 tcccaccact acagtatctc aacactccag caccccagca ccatagtgtt ccagcacccc 22920 agcatcccaa caccccagca cttaagcatc ccaacacctc ggcatcccaa caccccagca 22980 ctgcagcatc tcagcacctt agcatcccag tgccctagca tctcaatgct ccagcacacc 23040 agtactacag tattccagca ccccagcact ccagcatctc agcactgcag cactgcagca 23100 ctccagcatc ccaaaatccc agcatcccaa caccccagca gaccagcaga ccagcatctc 23160 agcaccgcag catccaagga ctatcccagc atcccagcaa cccagcacct cagcatccca 23220 acaccccagc atttcagcat ggcaacaccc cagtacccca gcacttcagc accccagtat 23280 cccagcatct cagcgaccca gtatcacaaa acctcagcat cctagcaccc cagcacccca 23340 gcaccttagc accttagcat cccagcatct cagcgcctca gcatcttgat attctggctg 23400 aggtcagcgt ggtgtatcta gtcagggtcc taactttcac ttcgcaggga aatgctgctg 23460 gactgggtct catgttgggc tgaagctctc tagacccctt gaagacagca taaaagagct 23520 tggagacgct gggtgtcccc catggaagag ttcactctca tcctgctttg acaacagcct 23580 tctctggggt ccctcacggg cccctctttc ttactgcaag tttgtctctg agaagactgt 23640 gatgcagaag tcactcagct gcctgtggct cctgaagagc tgaaggtgga ggcctgtagg 23700 cctccctatg agaggcgcag aaaaaaccat gattgctagt ggggaggtgc tccctctaca 23760 acccactcca taatctgccc ccgcccagct ctgaggccag ccccagggga aaatgccaga 23820 tccccaggga ggtgtgtgag acctcagggg ctccctcctc ccttacagca ggctcaggcc 23880 cctgggggcc tcagggccaa ggtctgtggg taagctacta tctctcactt gtcctctagc 23940 cacaaaagcc agggagatct ggcaatggac atgaggttct gaagaagcac atatgactgg 24000 cttcctaatg cgtggttgtt cagtgattca ataaacacgc atgggccagg catggggaaa 24060 tagacaaaca tgatccccaa cctctcccag agtgaactgg gagggaggag tgttcatccc 24120 tcaggattac accagagaaa caaaccagca ggagatatat atggttttgg ggggtcaaga 24180 aagaggaaaa acctggcaag gcaagtccaa aatcatagga caggctgtca ggaagggcag 24240 cctggaacct ctcaagcagg agctgatgct gcagtccaca ggcagaattt cttcttcctc 24300 ggggaaatct cagctttgtt cttaaggcct ttcaactgat tggctgaggt ctgccccttc 24360 ccccacattc tccaggataa tcttccttac ttaaagtcaa ctattaatca cagctacaaa 24420 atcccttcac agctacacat agatcagtgt ttgattgacg aacagcccct acagcctagc 24480 caagttgaca cataaaacta accatcacag ggggacaaat gatgtaaaca catcaacaaa 24540 taaaacagta acaagttaag gtctatggaa aaaacacaga aggggcagag agaaagaaag 24600 caagaaggag agtcccagtt tgctagggct tgtgggaagt ggggagcagt tctctttagc 24660 taggatattt gggaaaggca tatctgaagg agtgatattt gagcttagat taaaagatgg 24720 gaaggagcaa gccatgcaaa gagccaggat gttccaagca gagacggaac agcaagtgca 24780 aatgtcagga ggaatagaag gaggctggtg ggtggggtcc agtgagcaag aggagggcag 24840 gcaggagagg ggatggggag gtgggcaggc ccagaccacc cagggccctg gagactatcc 24900 tgatccaaca agggaagcct tgagtcactt cagtgtccat gtggagaatg gacctcagac 24960 tgaatgaggg aggcagtaag gagggcctct acctccaggg cttcgccctg tggactgcgc 25020 atagacatct ccaactcaga aagtctgaac caaactttcc atagttcccc caagtctggg 25080 catcctccta ctcagtgaaa ggcagccatc acacctccct gccctgctcc cggatgcccc 25140 aaatcctctt ggtctccaag tccagaacct gagacttgtc cttgatgttt gtctttccct 25200 caccctttct gtattctggg aagatgggtt tttttccccc agatgaatct gtaaaacttc 25260 tgtgatcaca ataaaaattc tggcagtatt attttctgga acatgacaaa gtgattcaaa 25320 attatttatc tggaagacta caaaacaaga atagccagga aatttctaaa aagaaagaag 25380 aaggaggagg agaaagaagg aggaggaaaa ggaggagaag aagaaaagaa aaagaaccaa 25440 gaaagggttc tagctctacc aaatattaaa acatatcatg aagctattta aaacaatatg 25500 gttgtggata ctgaaaaaga tgtgaataaa gtggaaggaa aataaataga aatgcacatg 25560 gggattgaga ctgtgaaaaa ggcagcatct cacatcagtg agggatgttc aacacctggt 25620 gttgggaaaa ctggctagtc atttaaacca aacaactggg tcctctacct cactcctgac 25680 attaagatac atttagatga ttcaaagagt aagacagaaa aaataacacg tgaaaacact 25740 atcagaaaac aacgtgggcc aggtgtggtg ggtcacgcct gtaatcccag cactttggga 25800 ggccgaggca gacagatcac ctgaggtggg gagttcaaga ccagcctgac caacatggtg 25860 aaatcctgtc tctactaaaa atacaaaatt agctgagcgt ggtggcgcat gcctgtaatc 25920 ccagctactc aggaggccga ggcaggagaa tcacttgaac ctgggaggca gaggttgtgg 25980 tgagccgaga tcacgccatt gcactccagc ctgggcaaca agagtgaaaa tccatctaaa 26040 aaaaaaaaaa aaagccaagg tggatatttt tatagtatca gggtagatca agcttctcca 26100 atcatgacat gaaacccaga aaccataaaa gaaaagaatg ataaaatcgc ccacgtaaag 26160 taaaaagctt gcacacagaa aaacaccata caggttacaa gatgagcagc aaaatcagag 26220 aaaaaacatt gcaattcagg acacacagag gctattgttc ctaatattta aaaataaaag 26280 tagtggattg tctacaaaaa gatgaagaca agaatttcag aaaaccaaat actgcatgtt 26340 ttcacttaca agtggaagct aaacactgag tacacgtgta cacaaagaat ggaaccatag 26400 gccaggcacc gtggctcacg cctgtaatcc cagtactttg cgaggccgaa gcgggcggat 26460 cacctgaggt gaggagttcg agaccatcct ggccaacatg gtgaaaccca gtctctacta 26S20 aaaatacaaa aattagccgg gcgtggtggt gggtgcctgt aatcccagct actcgggagg 26580 ctgcggcagt agaatcgctt gaaccctgga ggtggacctt gcagtgagcc gagatcgcac 26640 cactgcactc cagcctgggc aacagagtga gactccatct caaaaaaaaa aaaaaggaat 26700 agaacaatag acactggggc ctacttgagg gaggagggtg aggatcaaaa acctgcctat 26760 caggtactat gcttattacc tgggtggtga aataatctgt acaccaaacc ccagtgacat 26820 gcaatttacc gatgtaacaa acctgcccat gtacccgctg aacctaaaat aaaagttgga 26880 aaaaaatata gaaattttct ttgtaatagc caaaaactgc aaacagccca ggtgtctatt 26940 agtagaatgc ataaacaaac tcgggcatgt tcatacaatg taaaactact catcaataaa 27000 aagtgatact tctcagcaat gaaaagaaac tagctactga taccagctac aacatggatg 27060 gatttcaagt gctttatgat gagagcaaga agccagacac aaaagtgtct atatatatat 27120 acagtatata tacgtatata tacacatata tacagtatat atatacatat acatgtatat 27180 atatactgta tatatactgt atatatatac acagtatata tatacatata tacagtgtat 27240 atatactgtg tatatataca tgtatatata ctgtgtatat atacatgtat atatactgtg 27300 tatatataca tgtatatata ctgtgtatat atacatgtat atatatgtat actgtatata 27360 tactgtatat atatatacac atatatacag tatatatata cagtatatac tgtatatata 27420 cagtatatac gtgtatatat acatatatac agtatatatg taaatataca tatatacagt 27480 atatatgtaa atatacatat atacatgtat atatatacac tatatatata catatatagt 27540 gtatatatac atatatacat gtatatattt actatatgat tccatttata taaagtgcca 27600 aaacagtcaa aaataatcta tgtggaaaaa atcaacaaag ggatcccccg ggctgcagga 27660 attcgatggc gcgccgacgt cgcatgctcc tctagactcg aggaattcgg taccccgggt 27720 tcgaaatcga taagcttgga tccggagagc tcgataacat ttaaatggat ctaccacatt 27780 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 27840 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 27900 caatagcatc acaaatttca caaataaagc aatagcatca caaatttcac aaataaagca 27960 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 28020 gagctaggat cccgcggccg cctactggtc ctcctggagg tagaacttgg tgaccatgaa 28080 ggactctttg ggcttgttgg tgaggctgac gggccggtct gcctcctgcg ccgtgcagag 28140 gaaccagcca gggcaggcgg cagactcgaa gctggtggtg gggccactgt ttgagcggat 28200 gaaggtgaag cgcttgttct cctccttgtt cttgctcagg tcagtgatgt taactgcctc 28260 caattggaac ctaatctcat caccagactt gacacaggcc aggcacagct tcctcccatg 28320 gagtcccagg aatagagcat caggctcaat gggcaccaca tctatcttct cttgtaattt 28380 agtatttgat tcttgcaagt atccagcaac tagttggtta ttcetcatgt agaaggtctt 28440 ctggttaaca tcccagattc tgaaggcttg catcttgcag ggtctcttcc ccaaagggtg 28500 gcaggctgtc tctgagtaga gcaagaaaag gaggagagag attaggtgtc tgacagaacg 28560 cctgcggatt tccataccgg tggatccatg gctctgtctc aggtcagtat aggagctttg 28620 atgtgaagtc agccaagaac agctgaacac tacttcctct gaggcccttt tataggaggg 28680 attgcttcet gtgaataata ggaggatatt gtccacatcc agtaaagagg aaatccccaa 28740 ctgcatccaa aaagttttct gggaatatcc actgctgcag gagtggaaag tccccagtgg 28800 aaagtcccca gtggaaagtc cccagtggaa agtccccagt ggaaagtccc cagaatttcg 28860 acggcgcgcc taccagtaaa aaagaaaacc tattaaaaaa acaccactcg acacggcacc 28920 agctcaatca gtcacagtgt aaaaaagggc caagtgcaga gcgagtatat ataggactaa 28980 aaaatgacgt aacggttaaa gtccacaaaa aacacccaga aaaccgcacg cgaacctacg 29040 cccagaaacg aaagccaaaa aacccacaac ttcctcaaat cgtcacttcc gttttcccac 29100 gttacgtcac ttcccatttt aagaaaacta caattcccaa cacatacaag ttactccgcc 29160 ctaaaaccta cgtcacccgc cccgttccca cgccccgcgc cacgtcacaa actccacccc 29220 ctcattatca tattggcttc aatccaaaat aaggtatatt attgatgatg ttt 29273
REFERENCES CITED IN THE DESCRIPTION
This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be eæluded and the EPO disclaims all liability in this regard.
Patent documents cited in the description • US2Q030091536.A1 ίΟΡΡβ] • US5747072A Γ00081
Non-patent literature cited in the description • MATTHEWS, G.LHUNTER, D.J.Emerging drugs for osteoarthritisExpert Opin. Emerging Drugs, 2011, 1-1310002] • BROOKS PM.Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implicationsCurr Opin Rheumatol, 2002, vol. 14, 573-577 [08021 • Principals and practices of joint disease treatmentCARON JPGENOVESE, RLDiagnosis and Management of Lameness in the HorseSaunders20030000746-764 [00031 • lnterleukin-1 familyDINARELLO CAThe Cytokine HandbookAcademic Press20030000643-668 £00051 • EVANS, C.H.GOUZE, J.N.GOUZE, E.ROBBINS, P.D. GHIVIZZANI, S.C.Osteoarthritis gene therapyGene Ther, 2004, vol. 11,379-389 [00051 [00051 • CARON JP et al.Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expressionArthritis Rheum, 1996, vol. 39, 1535-1544 [QQOSj • Gene therapy for arthritisEVANS CHROBBINS PDGene Therapeutics: Methods and Applications of Direct Gene Transforml 9940000320-343 [0608] . D.D. FRISBIES.C. GHMZZANIP.D. ROBBINSC.H. EVANSC.W. MCLLWRAITHGene Ther, 2002, vol. 9, 12-20 Γ00661 • MITANI, K.GRAHAM, F.L CASKEY, C.T. KOCHANEK. S.Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vectorProc Natl Acad Sci USA, 1995, vol. 92, 3854-3858 [0007] • PARKS, R.J. et al.A Helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signalProc Natl Acad Sci USA, 1996, vol. 93, 13565-13570 [0007] • PARKS, R.J.Improvements in adenoviral vector technology: overcoming barriers for gene therapyClin. Genet., 2000, vol. 58, 1-1110007.1 • Multi-Year Transgene Expression in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral VectorsBRUNETTI-PIERRI, N. et al.Annual Meeting 2011 Molecular TherapyAmerican Society of Gene &amp; Cell Therapy20110500vol. 19, 0)607] • PALMER, D.NG, P.Improved system for helper-dependent adenoviral vector productionMol Ther, 2003, vol. 8, 846-852 Γ00171 100261 . TOIETTA, G.PASTORE, LCERULLO, V.FINEGOLD, M.BEAUDET, A.L LEE, B.Generation of helper-dependent adenoviral vectors by homologous recombinationMol Ther, 2002, vol. 5, 204-210 [0017].[0026] • SUZUKI, M.CELA, R.CLARKE, C.BERTIN, T.K.MOURINO, S.LEE, B.Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factoriesHum Gene Ther, 2010, vol. 21, 120-126 .[9026]. • RUAN, Z.DAWSON, B.JIANG M.M.GANNON, F.HEGGENESS, M.LEE, B.Quantitative volumetric imaging of murine osteoarthritic cartilage by phase contrast micro-computed tomography, 2012, [00301

Claims (16)

1. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem, omfattende en hjælperafhængig adenovirusvektor, der indeholder en nukleinsyre-sekvens, der koder for menneske- eller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra), L ITR og R ITR (left and right inverted terminal repeats), et adenoviralt pakkesignal og ikke-virale, ikke-kodende stuffernukle-insyresekvenser, hvor ekspressionen af menneske- eller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra)-genet reguleres af en inflammati-onsinducerbar promoter, der er lokaliseret opstrøms for læserammen af den nukleinsyresekvens, der koder for menneske- eller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra), og som aktiveres specifikt ved forhøjede niveauer af immunstimulerende stoffer.
2. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge krav 1, hvor den inflammationsinducerbare promoter er valgt fra gruppen bestående af NF-KB-promoter, interleukin-6 (ll-6)-promoter, interleukin-1 (11-1)-promoter, tumornekrosefaktor (TNF)-promoter, cyklooxygenase 2 (COX-2)-promoter, komplementfaktor 3 (C3)-promoter, serumamyloid A3 (SAA3)-promoter, makrofaginflammatorisk protein-1a (MlP-la)-promoter eller hybridkonstruktioner af ovennævnte.
3. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge krav 1 eller 2, hvor den hjælperafhængige adenovirusvektor omfatter en nukleinsyresekvens, der er angivet på side 15-30 eller 31 - 45 i beskrivelsen, eller en biologisk virksom del deraf.
4. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af de foregående krav, hvor pattedyrs-interieukin-1-receptorantagonisten (11-1 Ra) er valgt fra gruppen bestående af muse-ll-1Ra, heste-l 1-1 Ra, hunde-ll-1Ra, katte-11-1 Ra, kanin-l 1-1 Ra, hamster-ll-1 Ra, okse-ll-1Ra, kamel-ll-1Ra eller homologerne deraf i andre pattedyrsarter.
5. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af de foregående krav, hvor den hjælperafhængige adenovirusvektor endvi- dere omfatter et markørgen, der muliggør overvågning af vektorgenomet.
6. Adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af de foregående krav, hvor den hjælperafhængige vektor omfatter en nukle-insyresekvens, der er angivet på side 14 i beskrivelsen, eller en konserveret sekvens deraf, der koder for de samme aminosyrer.
7. Adenovi rusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af de foregående krav, hvor den hjælperafhængige vektor har mindst 50 %, 60 %, 80 %, 90 % sekvenshomologi med nukleinsyresekvensen, der er angivet på side 14 i beskrivelsen.
8. Farmaceutisk sammensætning, omfattende en hjælperafhængig adenovi-rusvektor, der indeholder en nukleinsyresekvens, der koder for menneskeeller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra), L ITR og R ITR (left and right inverted terminal repeats), et adenoviralt pakkesignal og ikke-virale, ikke-kodende stuffernukleinsyresekvenser, hvor ekspressionen af menneskeeller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra)-genet reguleres af en inflammationsinducerbar promoter, der er lokaliseret opstrøms for læserammen af den nukleinsyresekvens, der koder for menneske- eller pattedyrs-interleukin-1-receptorantagonist (11-1 Ra), og som aktiveres specifikt ved forhøjede niveauer af immunstimulerende stoffer.
9. Farmaceutisk sammensætning ifølge krav 8, hvor den inflammationsindu-cerbare promoter er valgt fra gruppen bestående af NF-KB-promoter, inter-leukin-6 (ll-6)-promoter, interleukin-1 (ll-l)-promoter, tumornekrosefaktor (TNF)-promoter, cyklooxygenase 2 (COX-2)-promoter, komplementfaktor 3 (C3)-promoter, serumamyloid A3 (SAA3)-promoter, makrofaginflammatorisk protein-1 α (MIP-1 a)-promoter eller hybrid konstruktioner af ovennævnte.
10. Farmaceutisk sammensætning ifølge krav 8 eller 9, hvor den hjælperafhængige adenovirusvektor omfatter en nukleinsyresekvens, der er angivet på side 15-30 eller 31 - 45 i beskrivelsen, eller en biologisk virksom del deraf.
11. Farmaceutisk sammensætning ifølge et af de foregående krav, hvor pattedyrs-interieukin-1-receptorantagonisten (11-1 Ra) er valgt fra grup- pen bestående af muse-ll-1Ra, heste-ll-1 Ra, hunde-ll-1 Ra, katte-ll-1 Ra, ka-nin-ll-1 Ra, hamster-ll-1 Ra, okse-ll-1Ra, kamel-ll-1Ra eller homologerne deraf i andre pattedyrsarter.
12. Farmaceutisk sammensætning ifølge et af de foregående krav, hvor den hjælperafhængige adenovirusvektor endvidere omfatter et markørgen, der muliggør overvågning af vektorgenomet.
13. Farmaceutisk sammensætning ifølge et af de foregående krav, hvor den hjælperafhængige vektor omfatter en nukleinsyresekvens, der er angivet på side 14 i beskrivelsen, eller en konserveret sekvens deraf, der koder for de samme aminosyrer.
14. Farmaceutisk sammensætning ifølge et af de foregående krav, hvor den hjælperafhængige vektor har mindst 50 %, 60 %, 80 %, 90 % se-kvenshomologi med nukleinsyresekvensen, der er angivet på side 14 i beskrivelsen.
15. Anvendelse af et adenovirusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af kravene 1 til 7 til ekspression af interleukin-1-receptorantagonist (11-1 Ra) i synoviale celler ex vivo.
16. Adenovi rusbaseret biologisk afgivelses- og ekspressionssystem ifølge et af kravene 1 til 7 til anvendelse ved behandling eller forebyggelse af osteo-artrose eller en osteoartrosetilstand i menneske- eller pattedyrsled.
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